CA2759093C - Corn event mir162 - Google Patents

Abstract

A novel transgenic corn event designated MIR162 is disclosed. The invention relates to nucleic acids that are unique to event MIR162 and to methods for detecting the presence of the MIR162 event based on DNA sequences of the recombinant constructs inserted into the corn genome that resulted in the MIR162 event and of genomic sequences flanking the insertion site. The invention further relates to corn plants comprising the transgenic genotype of MIR162 and to methods for producing a corn plant by crossing a corn plant comprising the MIR162 genotype with itself or another corn variety. Seeds of corn plants comprising the MIR162 genotype are also objects of the present invention. The invention also relates to methods of controlling insects using MIR162 corn plants.

Description

Corn Event MIR162 This is a division of Canadian Patent Application Serial No. 2,653,992 filed on May 24, 2007.
It is to be understood that the expression "the present invention" or the like used in this specification encompasses not only the subject-matter of this divisional application but that of the parent also.
BACKGROUND
[0001] The present invention relates generally to the field of plant molecular biology, plant transformation, and plant breeding. More specifically, the invention relates to insect resistant transgenic corn plants comprising a novel transgenic genotype and to methods of detecting the presence of nucleic acids that are unique to the transgenic corn plants in a sample and compositions thereof.

[0002] Plant pests are a major factor in the loss of the world's important agricultural crops. About $8 billion are lost every year in the U.S. alone due to infestations of non-mammalian pests including insects. In addition to losses in field crops, insect pests are also a burden to vegetable and fruit growers, to producers of ornamental flowers, and to home gardeners.

[00031 Insect pests are mainly controlled by intensive applications of chemical pesticides, which are active through inhibition of insect growth, prevention of insect feeding or reproduction, or cause death. Good insect control can thus be reached, but these chemicals can sometimes also affect other, beneficial insects. Another problem resulting from the wide use of chemical pesticides is the appearance of resistant insect varieties.
This has been partially alleviated by various resistance management praotices, but there is an increasing need for alternative pest control agents. Biological pest control agents, such.
. as Bacillus thuringiensis(Bt) strains expressing pesticidal toxins like 8-endotoxins, have also been applied to crop plants with satisfactory results, offering in alternative or compliment to chemical pesticides. The genes coding for some of these 5.-endotoxins have been isolated and their expression. iii heterologous hosts have been shown to provide another tool for the control of economically important insect pests. In particular, the expressiort.of Bt 6-endotoxins has provided efficient protection against selected insect pests, and transgenie plants expressing such toxins have been commercialized, allowing farmers to reduce applications of chemical insect control agents.
[00041 Another family of insecticidal proteins produced by Bacillus species during the vegetative stage of growth (vegetative insecticidal proteins (Vip)) has also been identified. U.S. Patents 5,877,012, 6,107,279, and 6,137,033 la ¨J506-87 describe a new class of insecticidal proteins called Vip3. Other disclosures, including WO 98/18932, WO 98/33991, WO 98/00546, and WO 99/57282, have also now identified homologues of the Vip3 class of proteins. Vip3 coding sequences encode approximately 88 lcDa proteins that possess insecticidal activity against a wide spectrum of lepidopteran pests, including, but not limited to, black cutworm (BCW, Agrotis ipsilon), fall armyworm (FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliot his virescens), sugarcane borer, (SCB, Diatraea saccharalis), lesser cornstalk borer (LCB, Elasmopalpus lignosellus), and corn earworm (CEW, Helicoverpa zea), and when expressed in transgenic plants, for example corn (Zea mays), confer protection to the plant from insect feeding damage.
[0005] Present plant transformation methods generally lead to the random integration of transgenes like vip3 into a host-plant genome. This random insertion of introduced DNA =
into the plant's genome can be lethal if the foreign DNA happens to insert into, and thus mutate, a critically important native gene. In addition, even if a random insertion event does not impair the functioning of a host cell gene, the expression of an inserted foreign gene may be influenced by "position effects" caused by the surrounding genomic DNA.
In some cases, the gene is inserted into sites where the position effects are strong enough to prevent the synthesis of an effective amount of product from the introduced gene. For example, it has been observed in plants that there may be wide variations in levels of expression of a heterologous gene introduced into a plant's chromosome among =
individually selected events. There may also be differences in spatial or temporal patterns of expression, for example, differences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory elements present in the introduced gene construct. In other instances, overproduction of the gene product has deleterious effects on the cell.
Because of these potential problems, it is common to produce hundreds of different events and screen those events for a single event that has desired transgene expression patterns and levels for commercial purposes. However, once a commercially viable site within the plant's .
genome is identified it would be advantageous to target genes of interest to that non-detrimental site.
[0006] Several methods for the targeted insertion of a nucleotide sequence of interest into a specific chromosomal site within a plant cell have been described. Site-specific recombination systems have been identified in several prokaryotic and lower eukaryotic organisms. Such systems typically comprise one or more proteins that recognize two copies of a specific nucleotide sequence, cleave and ligate those nucleotide sequences, and thereby provide a precise, site-specific exchange of genetic information.
Several site-specific recoMbinases are known in the art. These include, but are not limited to, e.g., the =
bacteriophage P1 Cre/lox system (Austin etal. (1981) Cell 25: 729-736), the R/RS .
recombinase system from the pSR1 plasmid of the yeast Zygosaccharo myces rozaii (Araki et at. (1985) J. Mol. Biol. 182: 191-203), the Gin/gix system of phage Mu (Maeser and Kahlmann (1991) Mol. Gen. Genet. 230: 170-176), the FLP/FRT recombinase system =
from the 2 µm plasmid of the yeast Saccharomyces cerevisiae (Broach et at.
(1982) Cell 29: 227-234), and the Int recombinase from bacteriophage Lambda (Landy (1989) Annu. Rev. Biochem. 58: 912-949; Landy (1993) CUM Opin. Genet. Bev. 3: 699-707;
Lorbach et at. (2000) J. Mol. Biol. 296: 1175-1181; and WO 01/16345). One particularly useful site-specific targeting approach, disclosed in US Patent Application Publication No. 2006/0130179, uses lambda integrase mediated recombination. The method comprises introducing into a plant cell a target nucleotide sequence comprising a first Integrase Recognition Site; introducing into the plant cell a donor nucleotide sequence comprising a second Integrase Recognition Site; and introducing into the plant cell an Integrase or Integrase complex. Another useful site-specific targeting approach is disclosed in US Patent Application Publication No.
2006/0253918, which uses homologous recombination to integrate one or more genes (gene stacking) at specific locations in the genome.
[0007] An event that has desired levels or patterns of transgene expression is useful for introgressing the transgene into other genetic backgrounds by sexual out-crossing using conventional breeding methods. Progeny of such crosses maintain the transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions. It would also be advantageous to be able to detect the presence of a particular event in order to determine whether progeny of a sexual cross contain a transgene of interest. In addition, a method for detecting a particular event would be helpful for complying with regulations requiring the pre-market approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well-known nucleic acid detection method including but not limited to thermal amplification (polymerase chain reaction (PCR)) using polynucleotide primers or DNA hybridization using nucleic acid probes. Typically, for the sake of simplicity and uniformity of reagents and methodologies for use in detecting a particular DNA
construct

3 that has been used for transforming various plant varieties, these detection methods generally focus on frequently used genetic elements, for example, promoters, terminators, and marker genes, because for many DNA constructs, the coding sequence region is interchangeable. As a result, such methods may not be useful for discriminating between constructs that differ only with reference to the coding sequence. In addition, such methods may not be useful for discriminating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA
adjacent to the inserted heterologous DNA ("flanking DNA") is known.
[00081 For the foregoing reasons, there is a need for insect resistant transgenic corn events comprising novel nucleic acid sequences which are unique to the transgenic corn event, useful for identifying the transgenic corn event and for detecting nucleic acids from the transgenic corn event in a biological sample, as well as kits comprising the reagents necessary for use in detecting these nucleic acids in a biological sample.
There is a further need to provide specific target sites within the maize genome to allow for targeting and control of insertion of nucleotide sequences to be integrated into the corn genome.
SUMMARY
100091 The present invention relates to a transformed corn (Zea mays) event, designated MIR162 comprising a novel transgenic genotype that comprises a vip3Aa20 coding sequence, which is unique to event MIR162. The vip3Aa20 coding sequence encodes a Vip3Aa20 insecticidal protein that confers insect resistance to MIR162 corn plants. The MIR162 event also comprises a pmi coding sequence encoding a PMI protein that confers upon corn cells the ability to utilize mannose as a carbon source. In addition to the vip3A20 coding sequence, the present invention also provides other nucleic acids that are unique to M1R162. The invention also provides transgenic corn plants comprising the nucleic acids unique to MIR162, seed from the transgenic corn plants, and to methods for producing a transgenic corn plant comprising the unique nucleic acids of the invention by crossing a corn inbred comprising the nucleic acids unique to MIR162 with itself or another corn line of a different genotype. An example of seed, and hence corn plants grown from the seed, comprising nucleic acids unique to MIR162 was deposited at the American Type Culture Collection as accession No. PTA-8166. The transgenic corn plants of the invention may have essentially all of the morphological and physiological characteristics of corresponding isogenic non-transgenic corn plants in addition to those

4 conferred upon the corn plants by the novel genotype of the invention.
Biological samples and extracts from MIR 162 corn plants, tissues and seeds are also provided by the present invention. The present invention also provides compositions and methods for detecting the presence of nucleic acids unique to MIR162 in biological samples based on the DNA
sequence of the recombinant expression cassettes inserted into the corn genome that resulted in the MIR162 event and of genomic sequences flanking the insertion site. The present invention also provides a non-detrimental insertion target site on a maize chromosome useful for inserting genes of interest to a specific location on the chromosome and to methods of altering a maize genome by inserting heterologous nucleic acids at the disclosed insertion site or in the vicinity of the disclosed insertion site. The MIR162 event can be further characterized by analyzing expression levels of the Vip3Aa20 and PMI proteins as well as by testing MIR162 for efficacy against lepidopteran insect pests. The present invention also provides methods of producing transgenic corn plants resistant to a broader spectrum of insect pests by stacking the Vip3Aa20 insect resistant trait with insect resistance traits different than Vip3Aa20.
[0010] The foregoing and other aspects of the invention will become more apparent from the following detailed description.
[0010a] In one particular embodiment, the present invention relates to a maize chromosomal target site located within a maize plant cell, on chromosome 5, wherein said target site is flanked 5' by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked 3' by nucleotides 25,513 to 45,513 of SEQ ID NO: 106, wherein the target site comprises a heterologous nucleic acid comprising a gene of interest.
[0010b] In another particular embodiment, the present invention relates to a method of making a transgenic maize plant comprising inserting a heterologous nucleic acid on chromosome 5, wherein said inserted heterologous nucleic acid is flanked 5' by nucleotides

5,454 to 25,454 of SEQ ID NO: 106 and flanked 3' by nucleotides 25,513 to 45,513 of SEQ
ID NO: 106.

DESCRIPTION OF THE SEQUENCES IN THE SEQUENCE LISTING
SEQ ID NO: 1 is the Vip3Aa20 coding sequence in MIR162.
SEQ ID NO: 2 is the Vip3Aa20 amino acid sequence.
SEQ ID NO: 3 is the sequence of plasmid pNOV1300.
SEQ ID Nos: 4-12 are primers and probes useful in a TAQMAN assay.
SEQ ID NO: 13 is the sequence of a vip3Aa20 probe.
SEQ ID NO: 14 is the sequence of a pmi probe.
SEQ ID Nos: 15-37 are primers useful in the present invention.
SEQ ID NO: 38 is the sequence of a vip3Aa20 amplicon.
SEQ ID Nos: 39-40 are primers useful in the present invention.
SEQ ID NO: 41 is the sequence of the CJ134/179 5' amplicon.
SEQ ID Nos: 42-43 are primers useful in the present invention.
SEQ ID NO: 44 is a vip3Aa20 3' amplicon.
SEQ ID NO: 45 is the 5' genome-insert junction.
5a SEQ ID NO: 46 is corn genome sequence flanking 5' to insert.
SEQ ID NO: 47 is the 3' insert-genome junction.
SEQ ID NO: 48 is corn genome flanking 3' to insert.
SEQ ID NO: 49 is the MIR162 insert and flanking sequences.
SEQ ID Nos. 50-54 are primers useful in the present invention.
SEQ ID NO: 55 is a 5' PCR amplicon SEQ ID Nos. 56-58 are primers useful in the present invention.
SEQ ID NO: 59 is a 3' PCR amplicon.
SEQ ID Nos. 60-105 are primers useful in the present invention.
SEQ ID NO: 106 is the sequence of the region of maize chromosome 5 comprising the disclosed chromosomal target site.
SEQ ID NO: 107 is the maize genomic sequence that was displaced by the insertion of heterologous DNA in MIR162.
DETAILED DESCRIPTION
100111 The following definitions and methods are provided to better define the present invention and to guide those of ordinary skill in the art in the practice of the present invention. Unless otherwise noted, terms used herein are to be understood according to conventional usage by those of ordinary skill in the relevant art. Definitions of common terms in molecular biology may also be found in Rieger el al., Glossary of Genetics:
Classical and Molecular, 5th edition, Springer-Verlag: New York, 1994. The nomenclature for DNA bases and amino acids as set forth in 37 C.F.R. 1.822 is used herein.
[00121 As used herein, the term "amplified" means the construction of multiple copies of a nucleic acid molecule or multiple copies complementary to the nucleic acid molecule using at least one of the nucleic acid molecules as a template. Amplification systems include, but not limited to the polymerase chain reaction (PCR) system, ligase chain reaction (LCR) system, nucleic acid sequence based amplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicase systems, transcription-based amplification system (TAS), and strand displacement amplification (SDA). See, e.g., Diagnostic Molecular Microbiology: Principles and Applications, D. H. Persing et al., Ed., American

6 Society for Microbiology, Washington, D.C. (1993). The product of amplification is termed an amplicon.
100131 A "coding sequence" is a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA. Preferably the RNA is then translated in an organism to produce a protein.
=
[0014] As used herein, the term "corn" means Zea mays or maize and includes all plant varieties that can be bred with corn, including wild maize species.
100151 "Detection kit" as used herein refers to a kit of parts useful in detecting the presence or absence of DNA unique to MIRI62 plants in a sample, wherein the kit comprises nucleic acid probes and/or primers of the present invention, which hybridize specifically under high stringency conditions to a target DNA sequence, and other materials necessary to enable nucleic acid hybridization or amplification methods.
[0016] As used herein the term transgenic "event" refers to a recombinant plant produced by transformation and regeneration of a plant cell or tissue with heterologous DNA, for example, an expression cassette that includes a gene of interest. The term "event" refers to the original transformant and/or progeny of the transformant that include the heterologous DNA. The term "event" also refers to progeny produced by a sexual outcross between the transformant and another corn line. Even after repeated backcrossing to a recurrent parent, the inserted DNA and the flanking DNA from the transformed parent is present in the progeny of the cross at the same chromosomal location. The term "event" also refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA
including the transgene of interest as the result of a sexual cross of one parental line that includes the inserted DNA (e.g., the original transformant and progeny resulting from selfing) and a parental line that does not contain the inserted DNA. Normally, transformation of plant tissue produces multiple events, each of which represent insertion of a DNA construct into a different location in the genome of a plant cell.
Based on the expression of the transgene or other desirable characteristics, a particular event is selected. Thus, "event MIR162", "MIR162" or "MIRI62 event" may be used interchangeably.
100171 An insect resistant MIRI62 corn plant can be bred by first sexually crossing a first parental corn plant consisting of a corn plant grown from a transgenic MIR162 corn plant, such as a MIR162 corn plant grown from the seed deposited at the ATCC under accession

7 No. PTA-6188, and progeny thereof derived from transformation with the expression cassettes of the embodiments of the present invention that confers insect resistance, and a second parental corn plant that lacks insect resistance, thereby producing a plurality of first progeny plants; and then selecting a first progeny plant that is resistant to insects; and selfing the first progeny plant, thereby producing a plurality of second progeny plants;
and then selecting from the second progeny plants an insect resistant plant.
These steps can further include the back-crossing of the first insect resistant progeny plant or the second insect resistant progeny plant to the second parental corn plant or a third parental corn plant, thereby producing a corn plant that is resistant to insects.
[0018] "Expression cassette" as used herein means a nucleic acid molecule capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence. The expression cassette may also comprise sequences not necessary in the direct expression of a nucleotide sequence of interest but which are present due to convenient restriction sites for removal of the cassette from an expression vector. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Typically, however, the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation process known in the art. The expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, or organ, or stage of development. An expression cassette, or fragment thereof, can also be referred to as "inserted sequence" or "insertion sequence" when transformed into a plant.
[0019] A "gene" is a defined region that is located within a genome and that, besides the aforementioned coding sequence, may comprise other, primarily regulatory, nucleic acid sequences responsible for the control of the expression, that is to say the transcription and translation, of the coding portion. A gene may also comprise other 5' and 3' untranslated

8 sequences and termination sequences. Further elements that may be present are, for example, introns.
[0020] "Gene of interest" refers to any gene which, when transferred to a plant, confers upon the plant a desired characteristic such as antibiotic resistance, virus resistance, insect resistance, disease resistance, or resistance to other pests, herbicide tolerance, improved =
nutritional value, improved performance in an industrial process or altered reproductive capability.
[0021] "Genotype" as used herein is the genetic material inherited from parent corn plants not all of which is necessarily expressed in the descendant corn plants. The MIR162 genotype refers to the heterologous genetic material transformed into the genome of a plant as well as the genetic material flanking the inserted sequence.
[0022] A "heterologous" nucleic acid sequence is a nucleic acid sequence not naturally associated with a host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleic acid sequence.
[0023] A "homologous" nucleic acid sequence is a nucleic acid sequence naturally associated with a host cell into which it is introduced.
[0024] "Operably-linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one affects the function of the other. For example, a promoter is operably-linked with a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or functional RNA (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences in sense or antisense orientation can be operably-linked to regulatory sequences.
[0025] "Primers" as used herein are isolated nucleic acids that are annealed to a complimentary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then extended along the target DNA
strand by a polymerase, such as DNA polymerase. Primer pairs or sets can be used for amplification of a nucleic acid molecule, for example, by the polymerase chain reaction (PCR) or other conventional nucleic-acid amplification methods.
[0026] A "probe" is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, such as a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complimentary to a strand of a target nucleic acid, in the case of the present invention, to a strand of genomic DNA
from corn event MIR162. The DNA of MIR162 can be from a corn plant or from a sample that

9 includes DNA from MIR162. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
[0027] Primers and probes are generally between 10 and 15 nucleotides or more in length. Primers and probes can also be at least 20 nucleotides or more in length, or at least 25 nucleotides or more, or at least 30 nucleotides or more in length. Such primers and probes hybridize specifically to a target sequence under high stringency hybridization conditions. Primers and probes according to the present invention may have complete sequence complementarity with the target sequence, although probes differing from the target sequence and which retain the ability to hybridize to target sequences may be designed by conventional methods.
[0028] As used herein gene or trait "stacking" is combining desired traits into one transgenic line. Plant breeders stack transgenic traits by making crosses between parents that each have a desired trait and then identifying offspring that have both of these desired traits. Another way to stack genes is by transferring two or more genes into the cell nucleus of a plant at the same time during transformation. Another way to stack genes is by re-transforming a transgenic plant with another gene of interest. For example, gene stacking can be used to combine two different insect resistance traits, an insect resistance trait and a disease resistance trait, or a herbicide resistance trait. The use of a selectable marker in addition to a gene of interest would also be considered gene stacking.
[0029] "Stringent conditions" or "stringent hybridization conditions"
include reference to conditions under which a probe will hybridize to its target sequence, to a detectably greater degree than to other sequences. Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or wash conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing).
Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing). Longer =
sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratopy Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier: New York; and Current Protocols in Molecular Biology, Chapter 2, Ausubel etal., Eds., Greene Publishing and Wiley-lnterscience: New York (1995), and also Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual (5th Ed.
Cols Spring Harbor Laboratory, Cold Spring Harbor, NY).
[0030] Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution.
Generally, high stringency hybridization and wash conditions are selected to be about 5 C
lower than the thermal melting point (T,,) for the specific sequence at a defined ionic strength and pH. The Trn is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Typically, under high stringency conditions a probe will hybridize to its target subsequence, but to no other sequences.
[0031] An example of high stringency hybridization conditions for hybridization of complementary nucleic acids which have more than 100 complementary residues on a filter in a Southern or northern blot is 50% formamide with 1 mg of heparin at 42 C, with the hybridization being carried out overnight. An example of very high stringency wash conditions is 0.15M NaCI at 72 C for about 15 minutes. An example of high stringency wash conditions is a 0.2x SSC wash at 65 C for 15 minutes (see, Sambrook, infra, for a description of SSC buffer).
[0032] Exemplary hybridization conditions for the present invention include hybridization in 7% SDS, 0.25 M NaPO4 pH 7.2 at 67 C overnight, followed by two washings in 5% SDS, 0.20 M NaPat pH7.2 at 65 C for 30 minutes each wash, and two washings in 1% SDS, 0.20 M NaPO4pH7.2 at 65 C for 30 minutes each wash. An exemplary medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is lx SSC at 45 C for 15 minutes. An exemplary low stringency wash for a duplex of, e.g., more than 100 nucleotides, is 4-6x SSC at 40 C for 15 minutes.
[0033] For probes of about 10 to 50 nucleotides, high stringency conditions typically involve salt concentrations of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M
Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30 C. High stringency conditions can also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2x (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under high stringency conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.

[0034] The following are exemplary sets of hybridization/wash conditions that may be used to hybridize nucleotide sequences that are substantially identical to reference nucleotide sequences of the present invention: a reference nucleotide sequence preferably hybridizes to the reference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 2X SSC, 0.1% SDS at 50 C, more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C
with washing in 1X SSC, 0.1% SDS at 50 C, more desirably still in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 0.5X SSC, 0.1%
SDS
at 50 C, preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, mM EDTA
at 50 C with washing in 0.IX SSC, 0.1% SDS at 50 C, more preferably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO4, 1 mM EDTA at 50 C with washing in 0.1X
SSC, 0.1% SDS at 65 C. The sequences of the present invention may be detected using all the above conditions. For the purposes of defining the invention, the high stringency conditions are used.
100351 "Transformation" is a process for introducing heterologous nucleic acid into a host cell or organism. In particular, "transformation" means the stable integration of a DNA
molecule into the genome of an organism of interest.
[0036] "Transformed / transgenic / recombinant" refer to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
The nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule.
Such an extrachromosomal molecule can be auto-replicating. Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof. A "non-transformed", "non-transgenic", or "non-recombinant" host refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule. As used herein, "transgenic" refers to a plant, plant cell, or multitude of structured or unstructured plant cells having integrated, via well known techniques of genetic manipulation and gene insertion, a sequence of nucleic acid representing a gene of interest into the plant genome, and typically into a chromosome of a cell nucleus, mitochondria or other organelle containing chromosomes, at a locus different to, or in a number of copies greater than, that normally present in the native plant or plant cell. Transgenic plants result from the manipulation and insertion of such nucleic acid sequences, as opposed to naturally occurring mutations, to produce a non-naturally occurring plant or a plant with a non-naturally occurring genotype.

Techniques for transformation of plants and plant cells are well known in the art and may comprise for example electroporation, microinjection, Agrobacterium-mediated transformation, and ballistic transformation.
[0037] As used herein, the term "unique" to MIR162 means distinctively characteristic of MIR162. Therefore, nucleic acids unique to event MIR162 are not found in other non-MIR162 corn plants.
[0038] The "Vip3" class of proteins comprises, for example, Vip3Aa, Vip3Ab, Vip3Ac, Vip3Ad, Vip3Ae, VipAf, Vip3Ag, Vip3Ba, and Vip3Bb, and their homologues.
"Homologue" means that the indicated protein or polypeptide bears a defined relationship to other members of the Vip3 class of proteins. "Vip3Aa20" is a Vip3 homologue unique to event MIR162. It was generated by spontaneous mutations introduced into the maize-optimized vip3Aa19 gene comprised in pNOV1300 (SEQ ID NO: 3) during the plant transformation process.
[0039] This invention relates to a genetically improved line of corn that produces an insect control protein, Vip3Aa20, and a phosphomannose isomerase enzyme (PM1) that allows the plant to utilize mannose as a carbon source. The invention is particularly drawn to a transgenic corn event designated MIR162 comprising a novel genotype, as well as to compositions and methods for detecting nucleic acids unique to MIR162 in a biological sample. The invention is further drawn to corn plants comprising the MIR162 genotype, to transgenic seed from the corn plants, and to methods for producing a corn plant comprising the M1R162 genotype by crossing a corn inbred comprising the genotype with itself or another corn line. Corn plants comprising the MIR162 genotype of the invention are useful in controlling lepidopteran insect pests including, but not limited to, black cutworm (BCW, Agrotis ipsdon), fall arrnyworm (FAW, Spodoptera frugiperda), tobacco budworm (TBW, Heliothis virescens), sugarcane borer (SCB, Diatraea saccharalis), lesser cornstalk borer (LCB, Elasmopalpus lignosellus), corn earworm (CEW, Helicoverpa zea), and western bean cutworm (WBCW, Striacosta albicosta). The invention is further drawn to a method of protecting transgenic corn from feeding damage whereby stacking the insect resistance trait of M1R162 with a different insect resistance trait in the same transgenic plant results is a corn plant that is protected from feeding damage to a greater degree than the insect resistance traits alone.
[0040] In one embodiment, the present invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence that is unique to event MIR162.

. .
[0041] In another embodiment, the present invention encompasses an isolated nucleic acid molecule that links a heterologous DNA molecule inserted into the genome of MIR162 to genome DNA in MIRI62 comprising at least 10 or more (for example 15, 20, 25, 50 or more) contiguous nucleotides of the heterologous DNA molecule and at least 10 or more (for example 15, 20, 25, 50, or more) contiguous nucleotides of the genome DNA .
flanking the point of insertion of the heterologous DNA molecule. Also included are nucleotide sequences that comprise 10 or more nucleotides of contiguous insert sequence from event MIRI62 and at least one nucleotide of flanking DNA from event adjacent to the insert sequence. Such nucleotide sequences are unique to and diagnostic for event MIR162. Nucleic acid amplification or hybridization of genomic DNA
from MIRI62 produces an amplicon comprising such unique sequences and is diagnostic for event MIRI 62. In one aspect of this embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ
ID
NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof.
[0042] In another embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence which comprises at least one junction sequence of event MIR162, wherein a junction sequence spans the junction between a heterologous expression cassette inserted into the corn genome and DNA from the corn genome flanking the insertion site and is diagnostic for the event. In one aspect of this embodiment, the junction sequence is selected from the group consisting of SEQ
ID NO:
45, SEQ ID NO: 47, and the complements thereof.
[0043] In another embodiment, the present invention encompasses an isolated nucleic acid molecule linking a heterologous DNA molecule to the corn plant genome in event MIRI62 comprising a sequence of from about 11 to about 20 contiguous nucleotides selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 47, and the complements thereof.
[0044] In another embodiment, the invention encompasses an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of SEQ ID
NO: I, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof. In one aspect of this embodiment, the isolated nucleic acid molecule is comprised in a corn seed deposited at the American Type Culture Collection under the accession No. PTA-8166, or in plants grown from the seed.
i ., [0045] In one embodiment of the present invention, an amplicon comprising a nucleotide sequence unique to event MIR162 is provided. In one aspect of this embodiment, the nucleotide sequence is selected from the group consisting of SEQ ID NO: 1, SEQ
ID NO:
38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof.
[0046] In another embodiment, the present invention encompasses flanking sequence primers for detecting event MIR162. Such flanking sequence primers comprise a nucleotide sequence of at least 10-15 contiguous nucleotides from the 5' or the 3' flanking sequence. In one aspect of this embodiment, the contiguous nucleotides are selected from nucleotides 1-1088 (inclusive) of SEQ ID NO: 49 (arbitrarily designated herein as the 5' flanking sequence), or the complements thereof. In another aspect of this embodiment, the 5' flanking sequence primers are selected from the group consisting of SEQ
ID NO:
36, SEQ ID NO: 39, SEQ ID NO: 53, SEQ ID NOs: 68-80, and the complements thereof.
In another aspect of this embodiment, the contiguous nucleotides are selected from nucleotides 9391-10579 (inclusive) of SEQ ID NO: 49 (arbitrarily designated herein as the 3' flanking sequence), or the complements thereof. In yet another aspect of this embodiment, the 3' flanking sequence primers are selected from the group consisting of SEQ ID NO: 58, SEQ ID NOs: 97-105, and the complements thereof 100471 In still another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer that function together in the presence of a event MIR162 DNA
template in a sample to produce an amplicon diagnostic for event MIR162. In one aspect of this embodiment, the first primer and/or the second primer is chosen from SEQ ID NO:
1 or the compliment thereof. In another aspect of this embodiment, the first primer and/or the second primer is selected from the group consisting of SEQ ID NOs: 15-35, SEQ ID
NO: 37, SEQ ID NO: 42, and the complements thereof. In yet another aspect of this embodiment, the amplicon that is produced by the pair of primers comprises SEQ
ID NO:
1, SEQ ID NO: 38, SEQ ID NO: 44, or the complements thereof.
[0048] In another embodiment, the present invention encompasses a pair of polynucleotide primers comprising a first polynucleotide primer and a second polynucleotide primer which function together in the presence of a event MIR

template in a sample to produce an amplicon diagnostic for event MIR162, wherein the first primer is or is complementary to a corn plant genome sequence flanking the point of insertion of a heterologous DNA sequence inserted into the genome of event MIR162, and the second polynucleotide primer sequence is or is complementary to the heterologous DNA sequence inserted into the genome of event MIR162.
[0049] In one aspect of this embodiment, the first polynucleotide primer comprises at least 10 contiguous nucleotides from a nucleotide sequence selected from the group consisting of nucleotides 1-1088 of SEQ ID NO: 49, nucleotides 9391-10579 of SEQ ID -NO: 49, and the complements thereof. In a further aspect of this embodiment, the first primer is selected from the group consisting of SEQ ID NO: 36, SEQ ID NO: 39, SEQ ID
NO: 53, SEQ ID NO: 57, SEQ ID NOs: 68-72, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID

NOs: 97-105, and the complements thereof. In another aspect of this embodiment, the second polynucleotide primer comprises at least 10 contiguous nucleotides from position 1089-9390 of SEQ ID NO: 49, or complements thereof. In still a further aspect of this embodiment, the second polynucleotide primer is selected from the group consisting of SEQ ID NOs: 15-35, SEQ ID NO: 37, SEQ ID NO: 40, SEQ ID NOs: 50-52, SEQ ID
NOs: 54, SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 63, SEQ ID NO: 73, SEQ ID
NO: 82, SEQ ID NO: 96, and the complements thereof.
[0050] In another aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 36, and the second polynucleotide primer which is set forth in SEQ
ID NO: 37, function together in the presence of a event MIR162 DNA template in a sample to produce an amplicon diagnostic for event MIR162 as described in Example 4.
In one embodiment of this aspect, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 38.
[0051] In yet another aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 39, and the second polynucleotide primer, which is set forth in SEQ ID NO: 40, function together in the presence of a corn event MIR162 DNA
template in a sample to produce an amplicon diagnostic for the corn event MIR162 as described in Example 4. In one embodiment of this aspect, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 41.
[0052] In another aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 53, and the second polynucleotide primer, which is set forth in SEQ
ID NO: 54, function together in the presence of a corn event MIR162 DNA
template in a sample to produce an amplicon diagnostic for the corn event MIR162 as described in Example 5. In one embodiment, the amplicon comprises the nucleotide sequence set forth in SEQ ID NO: 55.

[00531 In a still a further aspect of this embodiment, the first polynucleotide primer, which is set forth in SEQ ID NO: 58, and the second polynucleotide primer, which is set forth in SEQ ID NO: 56, function together in the presence of a corn event template in a sample to produce an amplicon diagnostic for the corn event MIR162 as described in Example 5. In one embodiment, the amplicon comprises the nucleotide =
sequence set forth in SEQ ID NO: 59.
[0054] Of course, it is well within the skill in the art to obtain additional sequence further out into the genome sequence flanking either end of the inserted heterologous DNA
sequences for use as a primer sequence that can be used in such primer pairs for amplifying the sequences that are diagnostic for the MIR162 event. For the purposes of this disclosure, the phrase "further out into the genome sequence flanking either end of the inserted heterologous DNA sequences" refers specifically to a sequential movement away from the ends of the inserted heterologous DNA sequences, the points at which the inserted DNA sequences are adjacent to native genomic DNA sequence, and out into the genomic DNA of the particular chromosome into which the heterologous DNA
sequences were inserted. Preferably, a primer sequence corresponding to or complementary to a part of the insert sequence should prime the transcriptional extension of a nascent strand of DNA or RNA toward the nearest flanking sequence junction. Consequently, a primer sequence corresponding to or complementary to a part of the genomic flanking sequence should prime the transcriptional extension of a nascent strand of DNA or RNA
toward the nearest flanking sequence junction. A primer sequence can be, or can be complementary to, a heterologous DNA sequence inserted into the chromosome of the plant, or a genomic flanking sequence. One skilled in the art would readily recognize the benefit of whether a primer sequence would need to be, or would need to be complementary to, the sequence as set forth within the inserted heterologous DNA sequence or as set forth SEQ
ID NO:
38 depending upon the nature of the product desired to be obtained through the use of the nested set of primers intended for use in amplifying a particular flanking sequence containing the junction between the genomic DNA sequence and the inserted heterologous DNA sequence.
[0055] In another embodiment, the present invention encompasses an isolated insecticidal protein comprising SEQ ID NO: 2 and a nucleic acid molecule encoding SEQ ID
NO: 2.
In one aspect of this embodiment, the nucleic acid molecule is SEQ ID NO: I.
The present invention also encompasses a chimeric gene comprising a heterologous promoter operably linked to the nucleic acid molecule, and to recombinant vectors and host cells comprising the chimeric gene.
100561 In yet another embodiment, the present invention encompasses a method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 in a sample comprising corn nucleic acids, wherein the method comprises: (a) contacting the sample with a pair of polynucleotide primers that, when used in a nucleic acid amplification reaction with genomic DNA from event MIR162 produces an amplicon that is diagnostic for event MIRI62; (b) performing a nucleic acid amplification reaction, thereby producing the amplicon; and (c) detecting the amplicon. In one aspect of this embodiment, the amplicon comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ
ID
NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the compliments thereof.
10057] In another embodiment, the present invention encompasses a method of detecting the presence of a nucleic acid molecule that is unique to event MIR162 in a sample comprising corn nucleic acids, wherein the method comprises: (a) contacting the sample with a probe that hybridizes under high stringency conditions with genomic DNA
from event MIR162 and does not hybridize under high stringency conditions with DNA
from a control corn plant; (b) subjecting the sample and probe to high stringency hybridization conditions; and (c) detecting hybridization of the probe to the DNA. Detection can be by any means well known in the art including fluorescent, chemiluminescent, radiological, immunological, and the like. In the case in which hybridization is intended to be used as a means for amplification of a particular sequence to produce an amplicon which is diagnostic for the MIR162 event, the production and detection by any means well known in the art of the amplicon is intended to be indicative of the intended hybridization to the target sequence where one probe or primer is utilized, or sequences where two or more probes or primers are utilized. The term "biological sample" is intended to comprise a sample that contains or is suspected of containing a nucleic acid comprising from between five and ten nucleotides either side of the point at which one or the other of the two terminal ends of the inserted heterologous DNA sequence contacts the genomic DNA
sequence within the chromosome into which the heterologous DNA sequence was inserted, herein also known as the junction sequences. In addition, the junction sequence comprises as little as two nucleotides: those being the first nucleotide within the flanking genomic DNA adjacent to and covalently linked to the first nucleotide within the inserted heterologous DNA sequence. In one aspect of this embodiment, the probe comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO:
38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, and the complements thereof.
[0058) In yet another embodiment, the present invention encompasses a kit for the detection of nucleic acids that are unique to event MIR162 in biological sample. The kit includes at least one nucleic acid molecule of sufficient length of contiguous polynucleotides to function as a primer or probe in a nucleic acid detection method, and which upon amplification of or hybridization to a target nucleic acid sequence in a sample followed by detection of the amplicon or hybridization to the target sequence, are diagnostic for the presence of nucleic acid sequences unique to event MIR162 in the sample. The kit further includes other materials necessary to enable nucleic acid hybridization or amplification methods. In one aspect of this embodiment, a nucleic acid molecule contained in the kit comprises a nucleotide sequence from SEQ ID NO:
1 or SEQ ID NO: 49. In another aspect of this embodiment, the nucleic acid molecule is a primer selected from the group consisting of SEQ ID NOs: 15-37, SEQ ID NO: 39, SEQ
ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NOs: 50-54, SEQ ID NOs: 56-58, SEQ ID NOs: 60-105, and the complements thereof. In yet another aspect of this embodiment, the amplicon comprises SEQ ID NO: 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, SEQ ID NO: 59, or the complements thereof. A variety of detection methods can be used including, but not limited to TAQMAN (Perkin Elmer), thermal amplification, ligase chain reaction, southern hybridization, ELISA methods, and colorimetric and fluorescent detection methods. In particular the present invention provides for kits for detecting the presence of the target sequence, i.e., at least the vip3Aa20 sequence or a junction sequence, in a sample containing genomic nucleic acid from MIR162. The kit is comprised of at least one polynucleotide capable of binding to the target site or substantially adjacent to the target site and at least one means for detecting the binding of the polynucleotide to the target site. The detecting means can be fluorescent, chemiluminescent, colorimetric, or isotopic and can be coupled at least with immunological methods for detecting the binding. A kit is also envisioned which can detect the presence of the target site in a sample, i.e., at least the vip3Aa20 sequence or a junction sequence of MIR162, taking advantage of two or more polynucleotide sequences which together are capable of binding to nucleotide sequences adjacent to or within about 100 base pairs, or within about 200 base pairs, or within about 500 base pairs or within about 1000 base pairs of the iarget sequence and which can be extended toward each other to form an amplicon which contains at least the target site.
[0059] In another embodiment, the present invention encompasses a method of detecting Vip3Aa20 protein in a biological sample, the method comprising: (a) extracting protein from event MIR162 tissue; (b) assaying the extracted protein using an immunological method comprising antibody specific for the Vip3Aa20 protein produced by the event; and (c) detecting the binding of said antibody to the Vip3Aa20 protein.
[0060] In yet another embodiment, the present invention encompasses a biological sample derived from a event MIRI 62 corn plant, tissue, or seed, wherein the sample comprises a nucleotide sequence which is or is complementary to a sequence that is unique to event MIRI 62, and wherein the sequence is detectable in the sample using a nucleic acid amplification or nucleic acid hybridization method. In one aspect of this embodiment, the nucleotide sequence is or is complementary to SEQ ID NO: I, SEQ ID
NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 55, or SEQ ID NO: 59. In another aspect of this embodiment, the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn by-products.
[0061] In another embodiment, the present invention encompasses an extract of a biological sample derived from a MIR162 corn plant, tissue, or seed comprising a nucleotide sequence which is or is complementary to a sequence that is unique to MIR162. In one aspect of this embodiment, the sequence is detectable in the extract using a nucleic acid amplification or nucleic acid hybridization method. In another aspect of this embodiment, the sequence is or is complementary to SEQ ID NO: 1, SEQ ID
NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, or SEQ ID NO: 59. In yet another aspect of this embodiment, the sample is selected from the group consisting of corn flour, corn meal, corn syrup, corn oil, cornstarch, and cereals manufactured in whole or in part to contain corn by-products.
00621 Another embodiment of the present invention encompasses a corn plant, or parts thereof, and seed from a corn plant comprising the genotype of the transgenic event MIR162, wherein the genotype comprises a nucleotide sequence set forth in SEQ
ID NO:
= 1, SEQ ID NO: 38, SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:
49, SEQ ID NO: 55, SEQ ID NO: 59, or the complements thereof. One example of corn seed comprising the nucleic acid molecules of the invention was deposited 23 January 2007 and assigned the ATCC Accession No. PTA-8166. In one aspect of this embodiment, the corn plant is from the inbred corn lines CG5NA58, CG5NA58A, CO3ND97, CG5NA01, CG5NF22, CG4NU15, CG00685, CG00526, CG00716, NP904, NP911, NP948, NP934, NP982, NP991, NP993, NP2010, NP2013, NP2015, NP2017, NP2029, NP2031, NP2034, NP2045, NP2052, NP2138, NP2151, NP2166, NP2 161, NP2171, NP2174, NP2208, NP2213, NP2222, NP2275, NP2276, NP2316, BCTT609, AF031, NPH8431, 894, BUTT201, R327H, 2044BT, and 2070BT. One skilled in the art will recognize however, that the MIR162 genotype can be introgressed into any plant variety that can be bred with corn, including wild maize species, and thus the list of inbred lines of this embodiment are not meant to be limiting.
[0063] In another embodiment, the present invention encompasses a corn plant comprising at least a first and a second DNA sequence linked together to form a contiguous nucleotide sequence; wherein the first DNA sequence is within a junction sequence and comprises at least about 11 contiguous nucleotides selected from the group consisting of nucleotides 1079-1098 of SEQ ID NO: 49, nucleotides 9381-9400, and the complements thereof, wherein the second DNA sequence is within the heterologous insert DNA sequence set forth in SEQ ID NO: 49, and the complements thereof; and wherein the first and the second DNA sequences are useful as nucleotide primers or probes for detecting the presence of corn event MIR162 nucleic acid sequences in a biological sample. In one aspect of this embodiment, the nucleotide primers are used in a DNA
amplification method to amplify a target DNA sequence from template DNA
extracted from the corn plant and the corn plant is identifiable from other corn plants by the production of an ampl icon corresponding to a DNA sequence comprising SEQ ID
NO: 45 or SEQ ID NO: 47.
[0064] Corn plants of the invention can be further characterized in that simultaneously digesting the plant's genomic DNA with the restriction endonucleases Kpnl, EcoRV or Ncol results in an about a 8 kb, a 13 kb or 4.6 kb vip3Aa20 hybridizing band, respectively, using a vip3Aa20 probe under high stringency conditions.
Exemplified herein is a vip3Aa20 probe comprising the nucleotide sequence set forth in SEQ
ID NO:
13.
[0065] Corn plants of the invention can be further characterized in that digesting the plant's genomic DNA with the restriction endonuclease Acc65I or BamHI results in a single pmi hybridizing band using a pmi probe under high stringency conditions.
Exemplified herein is a pmi probe comprising the nucleotide sequence set forth in SEQ
ID NO: 14.

[0066] -- In one embodiment, the present invention provides a corn plant, wherein the MIR162 genotype confers upon the corn plant insect resistance or ability to utilize mannose as a carbon source, or both insect resistance and the ability to utilize mannose as a carbon source. In one aspect of this embodiment, the transgenic genotype conferring insect resistance upon the corn plant of the invention comprises a vip3Aa20 gene and the transgenic genotype conferring the ability to utilize mannose as a carbon source upon the maize plant of the invention comprises a pmi gene.
[00671 -- In yet another embodiment, the present invention provides a method for producing a corn plant resistant to lepidopteran pests comprising: (a) sexually crossing a first parent corn plant with a second parent corn plant, wherein said first or second parent corn plant comprises event MIR162 DNA, thereby producing a plurality of first generation progeny plants; (b) selecting a first generation progeny plant that is resistant to one or more lepidopteran pests; (c) selfing the first generation progeny plant, thereby producing a plurality of second generation progeny plants; and (d) selecting from the second generation progeny plants, a plant that is resistant to one or more lepidopteran pests;
wherein the second generation progeny plants comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 45, SEQ ID NO: 47, SEQ
ID
NO: 49, SEQ ID NO: 55, and SEQ ID NO: 59.
[0068] -- In another embodiment, the present invention provides a method of producing hybrid corn seeds comprising: (a) planting seeds of a first inbred corn line comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID
NO:
45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 55, and SEQ ID NO: 59, and seeds of a second inbred line having a different genotype; (b) cultivating corn plants resulting from said planting until time of flowering; (c) emasculating said flowers of plants of one of the corn inbred lines; (d) sexually crossing the two different inbred lines with each other; and (e) harvesting the hybrid seed produced thereby. In one aspect of this embodiment, the first inbred corn line provides the female parents. In another aspect of this embodiment, the first inbred corn line provides the male parents. The present invention also encompasses the hybrid seed produced by the embodied method and hybrid plants grown from the seed.
[00691 -- One skilled in the art will recognize that the transgenic genotype of MIR162 can be introgressed by breeding into other corn lines comprising different transgenic genotypes. For example, a MIR162 corn inbred can be crossed with a corn inbred comprising the transgenic genotype of the lepidopteran resistant Btll event (US Patent ..10506-87 Nos. 6,114,608 and 6,342,660). The resulting seed and progeny plants have the stacked insect resistance traits and the combined spectrum of activity of Cryl Ab and Vip3Aa20. Another trait stack encompassed by the present invention includes combining the MIR162 insect resistance trait and the MIR604 insect resistance trait (US Patent Application publication No. 2005/0216970, published September 29, 2005). The stacked traits in the resulting seed and progeny confer upon the plants an increased spectrum of activity;
i.e. the plants are active against both lepidopteran and coleopteran insect pests.
100701 Therefore, the present invention encompasses a method of protecting a transgenic = corn plant from feeding damage by one or more insect pests wherein the method comprises stacking in the same transgenic corn plant a Vip3Aa20 insect resistance trait with another insect resistance trait that is different from Vip3Aa20, whereby the stacked traits protect the corn plant against feeding damage by one or more insect pests to a greater degree than would be expected due to the insect resistance traits alone. In one aspect of this embodiment, the Vip3Aa20 insect resistance trait comprised in event MIR162 is stacked with the Cry3A055 insect resistance trait comprised in event in the same transgenic corn plant by sexually crossing event-MIR162 with event or by transforming the traits together into the same plant.
100711 Examples of other transgenic events which can be crossed with a MIR162. inbred include, the glyphosate tolerant GA2I event, the glyphosate tolerant/lepidopteran insect resistant M0N802 event, the lepidopteran resistant DBT418 event, the male sterile event MS3, the phosphinothricin tolerant event B16,.the lepidopteran insect resistant event .
MON 80100, the phosphinothricin tolerant events T14 and T25, the lepidopteran insect resistant event 176, and the coleopteran resistant event M0N863, all of which are known.
in the art. It will be further recognized that other combinations or stacks can be made with = the transgenic genotype of the invention and thus these examples should not be viewed as limiting.
[0072] One skilled in the art will also recognize that_transgenic corn seed comprising the MIR162 genotype can be treated with various seed-treatment chemicals, including = insecticides, to augment or syngergize the insecticidal activity of the Vip3Aa20 protein.
[0073] The subject invention discloses herein a specific site on chromosome 5 in the maize genome that is excellent for insertion of heterologous nucleic acids.
Also disclosed is a 5' molecular marker (oriie2; nucleotides 1680-3338 of SEQ ID NO: 106) and a 3' molecular marker (gag; nucleotides 43,275-45,086 of SEQ ID NO: 106) useful in ..10506-87 identifying the location of a targeting site on chromosome 5. Thus, the subject invention provides methods to introduce heterologous nucleic acids of interest into this pre-established target site or in the vicinity of this target site. The subject invention also encompasses a corn seed and/or a corn plant comprising any heterologous nucleotide sequence inserted at the disclosed target site or in the general vicinity of such site. One option to accomplish such targeted integration is to substitute a different insert in place of the vip3Aa20 expression cassette exemplified herein. In this general regard, targeted homologous recombination, for example without limitation, can be used according to the.
subject invention. "Homologous recombination" refers to a reaction between any pair of nucleotide sequences having corresponding sites containing a similar nucleotide sequence (i.e., homologous sequences) through which the two molecules can interact (recombine) to form a new, recombinant DNA sequence. The sites of similar nucleotide sequence are each referred to herein as a "homology sequence". Generally, the frequency of homologous recombination increases as the length of the homology sequence increases.
Thus, while homologous recombination can occur between two nucleotide sequences that are less than identical, the recombination frequency (or efficiency) declines as the divergence between the two sequences increases. Recombination may be accomplished using one homology sequence on each of the donor and target molecules, thereby generating a "single-crossover" recombination product. Alternatively, two homology sequences may be placed on each of the target and donor nucleotide sequences.
Recombination between two homology sequences on the donor with two homology sequences on the target generates a "double-crossover" recombination product.
If the homology sequences on the donor molecule flank a sequence that is to be manipulated (e.g., a sequence of interest), the double-crossover recombination with the target molecule will result in a recombination product wherein the sequence of interest replaces a DNA
sequence that was originally between the homology sequences on the target molecule.
The exchange of DNA sequence between the target and donor through a double-crossover recombi_nation event is termed "sequence replacement." This type of technology is the subject of, for example, US patent Application Publication No. 2006/0253918.
With the disclosed target site now being identified and with the sequences surrounding the identified target site, the skilled person will recognize that other methods for targeted integration of heterologous nucleic acids may be used. Such = methods, for example without limitation, are disclosed in US Patent Application Publication No. 2007/0039074 and US Patent Application Publication No.
2006/0130179.

[0074] In one embodiment, the present invention encompasses a maize chromosomal target site located on chromosome 5 between a opie2 molecular marker set forth as nucleotides 1680-3338 of SEQ ID NO: 106 and a gag molecular marker set forth as nucleotides 43,275-45,086 of SEQ ID NO: 106, wherein the target site comprises a heterologous nucleic acid. In another embodiment, the maize chromosomal huge/
site is located on chromosome 5 between nucleotides 25,454 and 25,513 of SEQ ID NO:
106. In yet another embodiment, the chromosomal target site is flanked 5' by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked 3' by nucleotides 25,513 to 45,513 of SEQ
ID
NO: 106..
[0075] In one embodiment, the present invention encompasses a method of making a transgenic maize plant comprising inserting a heterologous nucleic acid at a position on chromosome 5 located between a opie2 molecular marker set forth as nucleotides 3338 of SEQ ID NO: 106 and a gag molecular marker set forth as nucleotides 43,275-45,086 of SEQ ID NO: 106. In another embodiment, the heterologous nucleic acid is inserted on chromosome 5 between nucleotides 25,454 and 25,513 of SEQ ID NO:
106.
In still another embodiment, the inserted heterologous nucleic acid is flanked 5' by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked 3' by nucleotides 25,513 to 45,513 of SEQ ID NO: 106 [0076] The transgenic genotype of the present invention can be introgressed in any corn inbred or hybrid using art recognized breeding techniques. The goal of plant breeding is to combine in a single variety or hybrid various desirable traits. For field crops, these traits may include resistance to insects and diseases, tolerance to herbicides, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant and ear height, is important.
[0077] Field crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is cross-pollinated if the pollen comes from a flower on a different plant.
[0078] Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny. A cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A
cross of two plants each heterozygous at a number of gene loci will produce a population of hybrid plants that differ genetically and will not be uniform.
[0079] Corn (Zea mays L.), can be bred by both self-pollination and cross-pollination techniques. Corn has separate male and female flowers on the same plant, located on the tassel and the ear, respectively. Natural pollination occurs in corn when wind blows pollen from the tassels to the silks that protrude from the tops of the ears.
[0080] A reliable method of controlling male fertility in plants offers the opportunity for improved plant breeding. This is especially true for development of corn hybrids, which relies upon some sort of male sterility system. There are several options for controlling male fertility available to breeders, such as: manual or mechanical emasculation (or detasseling), cytoplasmic male sterility, genetic male sterility, gametocides and the like.
[0081] Hybrid corn seed is typically produced by a male sterility system incorporating manual or mechanical detasseling. Alternate strips of two corn inbreds are planted in a field, and the pollen-bearing tassels are removed from one of the inbreds (female).
Providing that there is sufficient isolation from sources of foreign corn pollen, the ears of the detasseled inbred will be fertilized only from the other inbred (male), and the resulting seed is therefore hybrid and will form hybrid plants.
[0082] The laborious, and occasionally unreliable, detasseling process can be avoided by using one of many methods of conferring genetic male sterility in the art, each with its own benefits and drawbacks. These methods use a variety of approaches such as delivering into the plant a gene encoding a cytotoxic substance associated with a male tissue specific promoter or an antisense system in which a gene critical to fertility is identified and an antisense to that gene is inserted in the plant (see:
Fabinjanski, et al.
EPO 89/3010153.8 publication no. 329,308 and PCT application PCT/CA90/00037 published as WO 90/08828).
[0083] The use of male sterile inbreds is but one factor in the production of corn hybrids.
Plant breeding techniques known in the art and used in a corn plant breeding program include, but are not limited to, recurrent selection, backcrossing, pedigree breeding, restriction length polymorphism enhanced selection, genetic marker enhanced selection and transformation. The development of corn hybrids in a corn plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop inbred lines from breeding populations.
Corn plant breeding programs combine the genetic backgrounds from two or more inbred lines or various other germplasm sources into breeding pools from which new inbred lines are developed by selfing and selection of desired phenotypes. The new inbreds are crossed with other inbred lines and the hybrids from these crosses are evaluated to determine which of those have commercial potential. Plant breeding and hybrid development, as practiced in a corn plant-breeding program, are expensive and time-consuming processes.
[0084] Pedigree breeding starts with the crossing of two genotypes, each of which may have one or more desirable characteristics that is lacking in the other or which complements the other. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive generations. In the succeeding generations the heterozygous condition gives way to homogeneous lines as a result of self-pollination and selection. Typically in the pedigree method of breeding five or more generations of selfing and selection is practiced: F1 4 F2; F2 4 F3;
F3 4F4; F4 '4F.5; etc.
[0085] Recurrent selection breeding, backcrossing for example, can be used to improve an inbred line and a hybrid that is made using those inbreds. Backcrossing can be used to transfer a specific desirable trait from one inbred or source to an inbred that lacks that trait. This can be accomplished, for example, by first crossing a superior inbred (recurrent parent) to a donor inbred (non-recurrent parent), that carries the appropriate gene(s) for the trait in question. The progeny of this cross is then mated back to the superior recurrent parent followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. After five or more backcross generations with selection for the desired trait, the progeny will be homozygous for loci controlling the characteristic being transferred, but will be like the superior parent for essentially all other genes. The last backcross generation is then selfed to give pure breeding progeny for the gene(s) being transferred. A hybrid developed from inbreds containing the transferred gene(s) is essentially the same as a hybrid developed from the same inbreds without the transferred gene(s).
[0086] Elite inbred lines, that is, pure breeding, homozygous inbred lines, can also be used as starting materials for breeding or source populations from which to develop other inbred lines. These inbred lines derived from elite inbred lines can be developed using the pedigree breeding and recurrent selection breeding methods described earlier.
As an example, when backcross breeding is used to create these derived lines in a corn plant-breeding program, elite inbreds can be used as a parental line or starting material or source population and can serve as either the donor or recurrent parent.
[0087] A single cross corn hybrid results from the cross of two inbred lines, each of which has a genotype that complements the genotype of the other. The hybrid progeny of the first generation is designated Fi. In the development of commercial hybrids in a corn plant-breeding program, only the F1 hybrid plants are sought. Preferred F1 hybrids are more vigorous than their inbred parents. This hybrid vigor, or heterosis, can be manifested in many polygenic traits, including increased vegetative growth and increased yield.
[0088] The development of a corn hybrid in a corn plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, although different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrid progeny (F1). During the inbreeding process in corn, the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid progeny (F1). An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid between a defined pair of inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.
[0089] A single cross hybrid is produced when two inbred lines are crossed to produce the Fi progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A X B and C X D) and then the two F1 hybrids are crossed again (A X B) X (C X
D). A
three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A X B) and then the resulting Fi hybrid is crossed with the third inbred (A X
B) X C. Much of the hybrid vigor exhibited by F1 hybrids is lost in the next generation (F2). Consequently, seed from hybrids is not used for planting stock.
[0090] Hybrid seed production requires elimination or inactivation of pollen produced by the female parent. Incomplete removal or inactivation of the pollen provides the potential for self-pollination. This inadvertently self-pollinated seed may be unintentionally harvested and packaged with hybrid seed.

.30506-87 100911 Once the seed is planted, it is possible to identify and select these self-pollinated plants. These self-pollinated plants will be genetically equivalent to the female inbred line = used to produce the hybrid.
[00921 Typically these self-pollinated plants can be identified and selected due to their decreased vigor. Female selfs are identified by their less vigorous appearance for vegetative and/or reproductive characteristics, including shorter plant height, small ear size, ear and kernel shape, cob color, or other characteristics.
[00931 Identification of these self-pollinated lines can also be accomplished through molecular marker analyses. See, 'The Identification of Female Selfs in Hybrid Maize: A
. Comparison Using Electrophoresis and Morphology", Smith, J.
S. C. and Wych, R. D., Science and Technology 14, pp. 1-8 (1995). Through these technologies, the homozygosity of the self-pollinated line can be verified by analyzing allelic composition at various loci along the genorne. Those methods allow for rapid identification of the invention disclosed = herein. See also, "Identification of Atypical Plants in Hybrid Maize Seed by Postcontrol and Electrophoresis" Sarca, V. et al., Probleme de Genetica Teoritica si Aplicata Vol. 20 (1) p. 29-42.
[00941 As is readily apparent to one skilled in the art, the foregoing are only some of the = . various ways by which the inbred of the present invention can be obtained by those looking to introgress the transgenic genotype of the invention into other corn lines. Other means are available, and the above examples are illustrative only.
[00951 The following examples are intended solely to illustrate one or more preferred embodiments of the invention and are not to be construed as limiting the scope of the-invention.
EXAMPLES =
Example 1_ Transformatibn and Selection of the MIR162 Event .
.
[00961 The MIR604 event was produced by Agrobacterium-mediated transformation of a proprietary corn (Zea mays) line. Immature embryos were transformed essentially as .
described in Negrotto et al. (Plant Cell Reports 19:798-803, 2000), using a DNA
=
fragment from plasmid pNOV1300 (SEQ ID NO: 3).
..pNOVI300 contains a nucleotide sequence comprising tandem expression cassettes. The =

first expression cassette comprises a ZmUbiInt promoter region from a Zea mays polyubiquitin gene, which contains the first intron (GenBank Accession number S94464) operably linked to a vip3Aa19 coding sequence further operably linked to PEPC
Intron #9 from the phosphoenolpyruvate carboxylase gene (GenBank Accession Number X15239) from Zea mays (Matsuoka and Minami, 1989. European J. Of Biochem.
181:593-598) and a 35S terminator sequence from the 35S RNA from the cauliflower mosaic virus genome (Similar to GenBank Accession Number AF140604). Its function is to provide a polyadenylation sequence (Franck etal., 1980. Cell 21:285-294). The wP3Aa19 gene in pNOV1300 comprises a synthetic maize-optimized vip3Aa coding sequence (Estruch, et al., 1999.) which was synthesized to accommodate the preferred codon usage for maize (Murray et al., 1989). The synthetic vip3Aa19 coding sequence used in plant transformations encodes the identical amino acid sequence as the native vip3Aa 1 coding sequence found in the soil bacterium Bacillus thuringiensis strain AB88 (US Patent 5,877,012), with the exception of a single amino acid difference at position 284; the native viplial coding sequence encodes lysine, whereas the synthetic vip3Aa19 coding sequence encodes glutamine at this position. The vip3Aa19 coding sequence encodes an insect control protein, Vip3Aa19 that provides resistance to lepidopteran insects. The second expression cassette is comprised of a ZmUbilnt promoter operably linked to a pmi coding sequence (also known as E.coli manA) encoding phosphomannose isomerase (GenBank Accession number M15380), which catalyzes the isomerization of mannose-6-phosphate to fructose-6-phosphate (Negrotto etal., 2000). The pmi coding sequence is further operably linked to a nopaline synthase 3' end transcription termination and polyadenylation sequence.
[0097] Immature embryos were excised from 8 - 12 day old ears and rinsed with fresh medium in preparation for transformation. Embryos were mixed with the suspension of , Agrobacterium cells harboring the transformation vector pNOV1300, vortexed for seconds, and allowed to incubate for an additional 5 minutes. Excess solution containing Agrobacterium was aspirated and embryos were then moved to plates containing a non-selective culture medium. Embryos were co-cultured with the remaining Agrobacterium at 22 C for 2-3 days in the dark. Embryos were transferred to culture medium supplemented with ticarcillin (100 mg/ml) and silver nitrate (1.6 mg/1) and incubated in the dark for 10 days. Embryos producing embryogenic callus were transferred to cell culture medium containing mannose.

.30506-87 [0098] Regenerated plantlets were tested by TAQMAN PCR analysis (see Example 2) for the presence of both the pmi and vip3Aal 9 genes, as well as for the absence of the antibiotic resistance spectinomycin (spec) gene. It was later discovered (See Example 4 below) that during the transformation process two mutations were introduced into the vip3Aa19 coding sequence, one of which resuled in an amino acid change in the Vip3Aa19 protein. Therefore, this new vip3Aa coding sequence, which is unique to event MIR162, was designated vip3Aa20. The vip3Aa20 coding sequence encodes isoleucine at position 129 in place of the methionine residue encoded by the vip3Aa19 gene.
[0099] Plants positive for both transgenes, and negative for the spec gene, were transferred to the greenhouse for further propagation. Positive events were identified .and screened using insect bioassays against fall armyworm. Insecticidal events were characterized for copy number by TAQMAN analysis. MIR162 was chosen for further analysis based on having a single copy of the transgenes, good protein expression as identified by EL1SA, and good insecticidal activity against fall anriyworm.
. [00100] The breeding pedigree of the MIR162 event was as follows: To MIR162 plant (x NPH8431)-> NPH8431 (MIR162) F1 (x NP2161)->NP2161(MIR162) F1 (x NP2161)4NP2161 (MIR162).13C1F1 (x B9620)-->F1(x B9620)4BC1F1(x B9620)4BC2F1 (x B9620)-->BC3F1 (x B9620)->BC4F1 (x B9620). Plant material from the BC4 generation was used for the Southern analysis, copy number determination and sequencing of the insert DNA. Negative controls for the experiments consisted of 10 .
negative segrepnt plants from the BC4 generation.
=
Example.2. MIRI62 Detection by TAQMANPCR
[00101] TAQMAN analysis was essentially carried out as described in Ingham etal.
(Biotechniques, 31:132-140,2001). Briefly, genomic DNA was isolated fromi leaves of transgenic and non-transgenic corn plants using the Puregena Geriomic DNA Extraction kit (Gentra Systems, Minneapolis, MN) essentjally =
according to the manufacturer's instruction, except all steps were conducted in 1.2 ml 96-well plates. The dried DNA pellet was resuspended in TE buffer (10 Mm Tris-HCI, pH
8.0, 1mM EDTA).
1001021 TAQMAN PCR reactions were carried out in 96-well plates. For the endogenous.
corn gene control, primers and probes were designed specific to the Zea-mays alcohol WO 2007/142840 Per/1182007/012301 dehydrogenase (adhl) coding sequnce (Genbank accession no. AF044295). It will be recognized by the skilled person that other corn genes can be used as endogenous controls. Reactions were multiplexed to simultaneously amplify vip3Aa and adhl or pmi and adhL For each sample, a master mixture was generated by combining 20 1., extracted genomic DNA with 35 jiL 2x TAQMAN Univerial PCR Master Mix (Applied Biosystems) supplemented with primers to a final concentration of 900 nM each, probes to a final concentration of 100 nM each, and water to a 70 p.L final volume.
This mixture = was distributed into three replicates of 20 1_, each in 96-well amplification plates and sealed with optically clear heat seal film (Marsh Bio Products). PCR was run in the ABI
Prism 7700 instrument using the following amplification parameters: 2 min at 50 C and min at 95 C, followed by 35 cycles of 15 sat 95 C and 1 min at 60 C.
[00103] Results of the TAQMAN analysis demonstrated ,that event MIR162 had one copy of the vip3Aa20 gene and one copy of the pmi gene.
1001041 Primers and probes that were used in the TAQMAN PCR reactions are shown in Table 1.
Table 1. Primers used in TAQMAN Assay.
Primer Name Primer Sequence Sequence No:
Vip3Aa-forward 51CACCTTCAGCAACCCGAACTA3' SEQ ID NO: 4 Vip3Aa -reverse 5'GCTTAGCCTCCACGATCATCTT3' SEQ ID NO: 5 Vip3Aa -probe 5'GTCCTCGTCGCTGCCCTICACCT3' SEQ ID NO: 6 (5' label = FAM, 3' label = TAMRA) PMI-forward 5'CCGGGTGAATCAGCG1-1 13' SEQ ID NO: 7 PMI-reverse 51GCCGTGGCC1TTGACAGT3' SEQ ID NO: 8 PMI-probe 5'TGCCGCCAACGAATCACCGG3' SEQ ID NO: 9 (5' label = FAM, Mabel = TAMRA) ZmADH-267forward 5'GAACGTGTG1TGGGTTTGCAT3' SEQ ID NO: 10 ZmADH-337 reverse 5'TCCAGCAATCCITGCACCIT3' SEQ ID NO: 11 ZmADH-316 probe 5'TGCAGCCTAACCATGCGCAGGGTA3' SEQ ID NO: 12 (5 'label= TET, 3' label = TAMRA) Example 3. MIR162 Detection by Southern Blot = CA 02759093 2011-11-14 [001051 Genomic DNA used for southern analysis was isolated from pooled leaf tissue of plants representing the BC4 generation of M1R162 using essentially the method of Thomas etal. (Theor. Appl. Genet. 86:173-180, 1993).
All plants used for DNA isolation were individually analyzed using TAQMAN PCR
(as described in Example 2) to confirm the presence of a single copy of the vip3Aa20 gene and the pmi gene. For the negative segregant controls, DNA was isolated from pooled leaf tissue of negative segregants from the BC4 generation. These negative segregant plants were individually analyzed using TAQMAN PCR to confirm the absence of the vip3Aa20 and pmi genes, but were, as expected, positive for the endogenous maize adid = gene. =
[001061 Southern analysis was carried out using conventional molecular biology techniques. (See Chomczynski, P. 1992. Analytical Biochemistry 201:34-139) Genomic DNA (7.5 jig) was digested with restriction enzymes that digest within the event MIR162 insert, but not within the coding sequence that corresponds to the specific probe used in = the experiment. This approach allowed for determination of the number of copies of each gene, corresponding to the specific probe used for each Southern analysis, which was incorporated into event MIR162. =
[001071 Another series of restriction digests was performed in which the insert was digested with restriction enzymes that would release a fragment of known size from the = insert. This approach provided additional evidence for the presence of a single copy of each coding sequence present in MIRI 62 and allowed for the detection of partial copies of the insert that may be closely linked to the M1R162 insert. Following agarose gel electrophoresis and alkaline transfer to a Zeta-Probes GT membrane (Bio-Rad, Cat. No.
162-0195), hybridizations were carried out using full-length PCR generated elenient probes. The probes were labeled with 32P via random priming using the MegaPrimeTm system (Amersham Biosciences, Cat. No. RPN1607). Hybridization was carried out at 65 C, followed by multiple washes in 2X SSC, 0.1% SDS and then 0.1X.SSC and 0.1%
SDS. The membranes were then subjected to autoradiography.
1001081 Included in each Southern analysis were three control samples: (1) DNA
from a negative (non-transformed) segregant used to identify any endogenous Zea may:
sequences that may cross-hybridize with the element-specific probe; (2) DNA
from a negative segregant into which is introduced an amount of digested pNOV1300 that is equal to one copy number based on plasmid size was introduced, to demonstrate the sensitivity of the experiment in detecting a single gene copy within the Zea may: genome;

and (3) Digested pNOV1300 plasm id equal to one copy number based on plasmid size, to act as a positive control for hybridization as well as to demonstrate the sensitivity of the experiment.
[00109] The results of Southern analyses demonstrated that the MIR162 insert contains a single copy of the vip3Aa20 gene and pmi gene and contains no pNOV1300 backbone sequences. A vip3Aa19 probe (SEQ ID NO: 13) was used for the vip3Aa20 Southern analysis. The nucleotide sequences of vip3Aa19 and vip3Aa20 differ by two nucleotides and are 99.9 % identical. Therefore, the vip3Aa19 probe hybridized to the vip3Aa20 sequence present in MIR162 under stringent conditions. Using the vip3Aa19 probe, a Kpnl and an EcoRV digest resulted in single hybridization bands approximately 8 kb and 13 kb in size, respectively. In addition, an Ncof double digest resulted in a single hybridization band consistent with the expected size of 4.6 kb. Using the pmi probe (SEQ
ID NO: 14), a Acc651 and a BamHI digest resulted in single hybridization bands of approximately 4 kb and 6 kb in size, respectively. In addition, an Xmal +
HindIII double digest resulted in a single hybridization band consistent with the expected size of 8.1 kb.
The 8.1 kb Xmal + HindIII pNOV1300 band (positive control) also hybridized with the vip3Aa19 and pmi probes as expected. Some cross-hybridization in the plasmid-only lanes with the DNA ladder probe was detected. Typically commercially available DNA
ladders may contain some vector sequences that can cross-hybridize with the plasmid control sequences as observed in these experiments, but, this does not impact the findings of this study. Finally, a pNOV1300 backbone probe did not hybridize demonstrating the absence of incorporation of any pNOV1300 vector backbone sequences into MIR162 during the transformation process.
Example 4. Heterologous DNA Insert Sequencing [00110] The nucleotide sequence of the vip3Aa and pmi coding sequences in the heterologous DNA molecule inserted in MIR162 was determined to demonstrate overall integrity of the insert, contiguousness of the functional elements and to detect any individual basepair changes. The coding sequences were amplified from DNA
derived from the BC4 generation. PCR amplification was carried out using either Expand High Fidelity PCR system (Roche, Cat. No. 1732650) or PfuUltraTm Hotstart High-Fidelity DNA polymerase (Stratagene, Cat. No. 600390). Each PCR product was individually cloned into either pCle-XL-TOPO vector (Invitrogen, Cat. No. K4700-20) or pCle-BluntII-TOPO vector (Invitrogen, Cat. No. K2800-20) and three separate clones for each PCR product were identified and sequenced. Sequencing was carried out using the ABI3730XL analyzer using AB1 BigDye 1.1 or Big Dye 3.1 dGTP (for GC-rich templates) chemistry. The sequence analysis was done using the Phred, Phrap, and Consed package from the University of Washington and was carried out to an error rate of less than 1 in 10,000 bases (Ewing & Green, 1998. Genome Research 8:186-194). The final consensus sequence for each gene was determined by combining the sequence data , from the three individual clones to generate one consensus sequence for each gene.
Sequence alignment was performed using the ClustalW program with the following parameters: scoring matrix blosum55, gap opening penalty 15, gap extension penalty 6.66 (Thompson eta!, 1994. Nucleic Acids Research 22:4673-4680).
1001111 The full vip3Aa20 coding sequence was PCR amplified using primers MOV3Aa-01-5': 51ATGAACAAGAACAACACCAA3' (SEQ ID NO: 15) and MOV3Aa-01-3':
51CTACITGATGCTCACGTCGTAG3' (SEQ ID NO: 16) and PfuUltra Hotstart enzyme generating a 2370bp product. The PCR amplicon was sequenced using the primers shown in Table 2.
Table 2.
Primer Name Sequence (5'43') Sequence No.
b03503b ACGAGCAGAACCAGGTGC SEQ ID NO: 17 b03503c GGTGAAGAAGGACGGCAG SEQ ID NO: 18 b03503d ACCTGTCGCAAGCTGCTGGG SEQ ID NO: 19 b03503e TGGACAAGCTGCTGTGTC SEQ ID NO: 20 b03503f TGCAGGCCGACGAGAACAG SEQ ID NO: 21 b03503g TGATCCAGTACACCGTGAA SEQ ID NO: 22 b03503h ACCCTGACCCTGTACCAG SEQ ID NO: 23 b03504b GIGTTGCCGCTGATGTTG SEQ ID NO: 24 b03504c CGTACTCGGTCTIVGGCT SEQ ID NO: 25 b03504d CTGCAGGCCAAAGCCGTT SEQ ID NO: 26 b03504e TCGCCGTAGATCACCTCG SEQ ID NO: 27 b03504f GCTTGCGACAGGTGGTCA SEQ ID NO: 28 b03504g TTGCTGCTGGTCTCGGTGG SEQ ID NO: 29 b03504h CGTTGGCGATCTTAAGGAT SEQ ID NO: 30 b00203c GCAAGCCATCGATTCAC SEQ ID NO: 31 b00203d GCAACACCCTGACCCTG SEQ ID NO: 32 b00203e TCTACGACGTGAGCATCAAG SEQ ID NO: 33 b00203f GTAGAAGTGCACGATCGGG SEQ ID NO: 34 b00203g CGGTGCTGGTCCAGTTG SEQ ID NO: 35 [001121 Two other PCR reactions overlapped the full vip3Aa20 coding sequence.
The 5' end of vip3Aa20 was covered with a PCR amplification using primers 162INSERT-F2:
5'ACACCAATGATGCAAATAGGC3' (SEQ ID NO: 36) and VIP R4 5'GAAGGTGTTCAGGTAGAACTCGAAG3' (SEQ ID NO: 37) and Expand High Fidelity enzyme. The second reaction Covered the 3' end of vip3Aa20; the product was amplified with primers VIP-F3: 5'GGTGCTGTTCGAGAAGAGGT3' (SEQ ID NO: 42) and PMI_REV1: 5'CGA1TTATCACTCTCAATCACAT3' (SEQ ID NO: 43) and Expand High Fidelity enzyme. The amplicons generated by these reactions comprised a 2946 bp nucleotide sequence (SEQ ID NO: 38) and a 2577 bp nucleotide sequence (SEQ ID
NO:
44), respectively.
1001131 The consensus sequence data revealed two nucleotide changes in the vip3Aa coding sequence in MIRI62 (designated vip3Aa20) compared to the vip3Aa coding sequence in pNOV1300 (designated vip3Aa19), which was used to transform MIR162.
The first nucleotide change, a G to T mutation, occurred at position 387 of the vip3Aa19 coding sequence (SEQ ID NO: 3). This mutation resulted in the methionine at position 129 of Vip3Aa19 being changed to isoleucine in Vip3Aa20 (M129I). The second nucleotide change occurred at position 1683 of the coding sequence, a G to C
mutation, but did not result in an amino acid change. Therefore, the vO3Aa20 coding sequence and the Vip3Aa20 protein are unique to the MIR162 event and can be used to identify any plant comprising the MIR162 transgenic genotype. The pmi coding sequence was identical to that in the transformation plasmid pNOV1300. An alignment of the Vip3Aa20 and Vip3Aa19 insecticidal proteins is shown in Table 3.
Table 3. Comparison of Vip3Aa20 and Vip3Aa19 amino acid sequences.
Name Sequence Alignment Vip3Aa20 (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE
Vip3Aa19 (1) MNKNNTKLSTRALPSFIDYFNGIYGFATGIKDIMNMIFKTDTGGDLTLDE
Vip3Aa20 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL
Vip3Aa19 (51) ILKNQQLLNDISGKLDGVNGSLNDLIAQGNLNTELSKEILKIANEQNQVL
Vip3Aa20 (101) NDVNNKLDAINTMLRVYLPKITSMLSDtQNYALSLQIEYLSKQLQEIS
Vip3Aa19 (101) NDVNNKLDAINTMLRVYLPKITSMLSD QNYALSLQIEYLSKQLQEIS
Vip3Aa20 (151) DICLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG
Vip3Aa19 (151) DKLDIINVNVLINSTLTEITPAYQRIKYVNEKFEELTFATETSSKVKKDG
Vip3Aa20 (201) SPADILDELTELTELAKSVTKNDvDGFEFYLNTFHDVmVGNNLFGRSALK
Vip3Aa19 (201) SPADILDELTELTELAKSVTKNDVDGFEFYLNTFHDVMVGNNLFGRSALK
Vip3Aa20 (251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD

Vip3Aa19 (251) TASELITKENVKTSGSEVGNVYNFLIVLTALQAQAFLTLTTCRKLLGLAD
Vip3Aa20 (301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVKGSDEDAKMIVE
Vip3Aa19 (301) IDYTSIMNEHLNKEKEEFRVNILPTLSNTFSNPNYAKVMSDEDAKMIVE
Vip3Aa20 (351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL
Vip3Aa19 (351) AKPGHALIGFEISNDSITVLKVYEAKLKQNYQVDKDSLSEVIYGDMDKLL
Vip3Aa20 (401) CPDQSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI
Vip3Aa19 (401) CPDOSEQIYYTNNIVFPNEYVITKIDFTKKMKTLRYEVTANFYDSSTGEI
Vip3Aa20 (451) DLNKKKVESSEAEYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS
Vip3Aa19 (451) DLNKKKVESSEABYRTLSANDDGVYMPLGVISETFLTPINGFGLQADENS
Vip3Aa20 (501) RLITLTCKSYLRELLLATDLSNKETKLIVPPSGFISNIVENGSIEEDNLE
Vip3Aa19 (501) RLITLTCKSYLRELLLATDLSNKETKL/VPPSGFISNIVENGSIEEDNLE
Vip3Aa20 (551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT
Vip3Aa19 (551) PWKANNKNAYVDHTGGVNGTKALYVHKDGGISQFIGDKLKPKTEYVIQYT
Vip3Aa20 (601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK
Vip3Aa19 (601) VKGKPSIHLKDENTGYIHYEDTNNNLEDYQTINKRFTTGTDLKGVYLILK
Vip3Aa20 (651) SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY
Vip3Aa19 (651) SQNGDEAWGDNFIILEISPSEKLLSPELINTNNWTSTGSTNISGNTLTLY
Vip3Aa20 (701) QGGRGILKONLQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAK
Vip3Aa19 (701) QGGRGILKQNIQLDSFSTYRVYFSVSGDANVRIRNSREVLFEKRYMSGAK
Vip3Aa20 (751) DVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK
Vip3Aa19 (751) DVSEMFTTKFEKDNFYIELSQGNNLYGGPIVHFYDVSIK
The shaded box indicates the amino acid change.
Example 5. Analysis of Flanking DNA Sequence [00114] A number of methods are known to those of skill in the art to amplify unknown DNA sequences adjacent to a core region of known sequence. Those methods include, but are not limited to, inverse PCR (tPCR) [Ochman et. al, Genetics 120:621-623(1988);
Triglia et. al., Nucleic Acids Res. 16:8186 (1988)], panhandle PCR [Jones and Winistorfer, Nucleic Acids Res. 20:595-600 (1992); Jones and Winistorfer, Biotechniques 23:132-138(1997)1, cassette ligation-anchored PCR [Mueller and Wold, Science 246:780-786 (1989)], vectorette-PCR [Riley et. al., Nucleic Acids Res. 18:2887-(1990)], novel-Alu-PCR [Puskas et. al., Nucleic Acids Res. 22:3251-3252 (1994)] and Thermal Asymmetric Interlaced PCR (TAIL-PCR) [Liu and Whittier, Genomics 25:673-681 (1995)].
[00115] One method used to amplify corn genome DNA sequence flanking the heterologous DNA inserted into event MIR162 was vectorette PCR essentially as described by Riley et al., Nucleic Acids Res. 18:2887-2890 (1990).
1001161 The 5' flanking sequence and junction sequence was confirmed using standard PCR procedures. The following primer pairs, or complements thereof, were used to confirm the sequence: 162INSERT-F2: 5'ACACCAATGATGCAAATAGGC3' (SEQ ID
NO: 36)/ VIP R4: 5.GAAGGTGITCAGGTAGAACTCGAAG3' (SEQ ID NO: 37) and CJB179: 51ATGCAAATAGGCTGGGAATAGTC3 (SEQ ID NO: 39)/ C113134 5'GTACCAGCTTGCTGAGTGGCT3' (SEQ ID NO: 40). The resulting amplicon has the sequence shown is SEQ ID NO: 41 and comprises the 5' junction sequence of SEQ
ID
NO: 45. It will be recognized that other primer sequences can be used to confirm the -flanking and junction sequences. Using this method, the MIRI62 insert was found to be flanked 5' by nucleotides-1040-1088 of the corn genomic sequence shown in SEQ
ID NO:
46.
100117) A larger region of the 5' flanking sequence from event MIR162 was generated using the Seegene DNA Walking SpeedUpTh4 Premix kit following the manufacturer's instructions.
100118) A first PCR reaction was performed independently in four individual tubes using primer FE1002: 5'CGTGACTCCCTTAA1TCTCCGCT3' (SEQ ID NO: 50) with one of the DW-ACP 1,2, 3, or 4 primers supplied by the manufacturer. The following reagents were mixed in a PCR tube on ice: 100 jig MIR162 genomic DNA, 4 id 2.5 DW-ACP -(one each with DW-ACP 1,. 2, 3, or 4), 4 I 2.5 M FE1002, 19 id distilled water, and 25 p.1 2X SeeAmpTM ACPTM Master Mix II. The tubes were placed in a preheated (94 C) thermal cycler_ PCR was completed using the following program: one cycle at 94 C for five minutes, 42 C for one minute, and 72 C for two minutes, 30 cycles of 94 t for 40 seconds, 55 C for 40 seconds, and 72 C for 90 seconds, and one cycle at 72 C
for seven minutes. The PCR products were purified using Exonuciease I and Shrimp Alkaline Phosphatase. =
1001.19) A second PCR reaction was performed independently in four individual tubes = ______________________________________________ using prime,: FE1003:
5.GATCAGATTGTail 1CCCOCCTT3' (SEQ ID.NO: 51) with the DW-ACPN primer supplied by the manufacturer of the kit. The following reagents were mixed in a PCR tube on ice: 3 .1 purified PCR product, I p.110 M DW-ACPN, I
1 10 idyl FE1003, 5 I distilled water, and 10 Id 2X SeeAmpTM ACPTM Master Mix IL
The tubes were placed in a preheated (94 C) thermal cycler. PCR was completed using =
the following program: one cycle at 94 C for five minutes, 35 cycles of 94 C
for 40 -je seconds, 60 C for 40 seconds, and 72 C for 90 seconds, and one cycle at 72 C
for seven minutes.
1001201 A third PCR reaction was performed independently in four individual tubes using primer FE1004: 51GATTGTCG1TTCCCGCCTTCAGTT3' (SEQ ID NO: 52) with the Universal primer supplied by the manufacturer. The following reagents were mixed in a PCR tube on ice: 2 I purified PCR product, 1 I 10 M Universal primer, 1 p110 M
FE1004, 6 I distilled water, and 10 pi 2X SeeAmpTM ACPTM Master Mix II. The tubes were placed in a preheated (94 C) thermal cycler. PCR was completed using the following program: one cycle at 94 C for five minutes, 35 cycles of 94 C for 40 seconds, 60 C for 40 seconds, and 72 C for 90 seconds, and one cycle at 72 C for seven minutes.
[001211 Ten I of the PCR products were run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli, and the plasmid DNA was extracted from the cells after overnight growth with a Qiagen Miniprep kit according to the manufacturer's instructions. This plasmid was used for end run sequencing.
1001221 A new primer was designed within the new, previously unknown sequence to be used with a primer in the heterologous DNA insert to amplify the full 1 kb of flanking sequence out of the genomic DNA. Flanking sequence primer 162DWConf3:
51CCTGTGTTGTTGGAACAGAC1TCTGTC3' (SEQ ID NO: 53) and insert DNA
primer FE0900: 51GGCTCCITCAACGTCGCGGTTCTGTC3' (SEQ ID NO: 54) were used to amplify a nucleic acid molecule comprising the 5' flanking sequence for confirmation. The sequence of the resulting amplicon is set forth in SEQ ID
NO: 55. This 5' amplicon comprises the 5' junction sequence set forth in SEQ ID NO: 45. Ten I of the PCR product (amplicon) was run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coil. Plasmid DNA was extracted from the cells after overnight growth in media with a Qiagen Miniprep kit according to the manufacturer's instructions. Three plasm ids were completely sequenced using the primers shown in Table 4. The plasmid sequences were aligned to generate the complete confirmed 5' flanking sequence. Using this method, approximately I kb of the 5' flanking sequence (SEQ ID NO: 46) was determined.
Table 4. Primer sequences.
Primer Name Sequence (5'43') Sequence No.
b0020 I h TTCACGGGAGACTTTATCTG SEQ ID NO: 60 b00605a CCGATTCATTAATGCAG SEQ ID NO: 61 b00701b ACGTAAAACGGCTTGTC SEQ ID NO: 62 b00702b GTTTAAACTGAAGGCGG SEQ ID NO: 63 b00704h AATAATATCACTCTGTACATCC SEQ ID NO: 64 b01 106f GTTGTAAAACGACGG SEQ ID NO: 65 b01709f TAGGCACCCCAGGCTTTA SEQ ID NO: 66 b03504a AATTGAATTTAGCGGCCG SEQ ID NO: 67 b05102f GGTCCCTACAACATAAATAG SEQ ID NO: 68 b05102g TTCGTCCCTACTATCAACGC SEQ ID NO: 69 b05102h CTTTAGGCATCAGCGGGT SEQ ID NO: 70 b05103a AGCATCTGCGTAAGCACA SEQ ID NO: 71 b05103b CTGATGACACCAATGATGC SEQ ID NO: 72 b05103c GATCAGATTGTCGTTTCCC SEQ ID NO: 73 b05103d GCATCATTGGTGTCATCAG SEQ ID NO: 74 b05103e TGTGCTTACGCAGATGCT SEQ ID NO: 75 b05103f ACCCGCTGATGCCTAAAG SEQ ID NO: 76 b05103g GCGTTGATAGTAGGGACGAA SEQ ID NO: 77 b05103h CTATTTATGTTGTAGGGACC SEQ ID NO: 78 b05210a CTAGACTGGAAAGCGGAG SEQ ID NO: 79 b052 10b CCACTTTCATCCCTAGTTG SEQ ID NO: 80 [00123] The 3' flanking sequence from event MIR162 was generated using the Clonetech GenomeWalkerTm Universal kit (Clonetech Laboratories, Inc.) following the manufacturer's instructions.
[001241 First, pools of uncloned, adaptor-ligated genomic DNA fragments, known as Genome Walker "libraries" were constructed. Each library was constructed by digesting the MIR162 genomic DNA with a restriction enzyme (Di-al, EcoRV , Pvull, Stul, and Xmnl) as follows: For example, 25 I MIR162 genomic DNA (0.1 g/p.1), 8 I
restriction enzyme (10 units/ 1), 10 I restriction enzyme buffer (10X), and 57 I
distilled 1-120 were mixed in a tube and incubated at 37 C overnight.
[001251 DNA was then purified by using several rounds of phenol/ chloroform extraction.
Finally, the DNA was precipitated and washed with ethanol, dried and dissolved into 20 I of TE buffer.
100126] To ligate the GenomeWalker Adapter ends to the MIR162 genomic DNA, 4 pi of the digested, purified genomic DNA was mixed with 1.9 I of Genome Walker Adapter (25 M), 1.6 Id 10X Ligation Buffer, and 0.5 11 14 DNA Ligase (6 units/ 1).
These reactions were incubated overnight at 16 C. The reactions were stopped with incubation at 70 C for five minutes. After the reaction was stopped, 72 I of TE was added to each tube, and the contents were mixed thoroughly.
[00127] A first PCR reaction was performed using primer API, supplied by the manufacturer, with different primers designed within the known heterologous insert DNA
sequence (Round 1 "gene specific primers" or "GSP1"). The following reagents were mixed in a PCR tube on ice: I pl of the appropriate MIR162 DNA library, 1 p110 pM
API, I I 10 WA GSP I, 1 I 10 mM dNTPs, 5 I 10X Advantage 2 PCR Buffer, 1 I
of BD Advantage 2 Polymerase, and 40 I distilled water. PCR was completed using the following program: seven cycles at 94 C for 25 seconds and 72 C for four minutes, 32 cycles at 94 C for 25 seconds and 67 C for four minutes, and one cycle at 67 C
for four minutes. Each primary PCR reaction was diluted 50-fold by adding 1 1 of the primary PCR product with 49 pl of distilled water. The reactions that worked were (1) the Dral and the Xmnl libraries with the GSP1 primer 162GW3F1:
51TCTCTTGCTAAGCTGG4JAGCTCGATCCG3' (SEQ ID NO: 56) and primer API.
[00128] A second PCR reaction was performed independently using primer AP2, supplied by the manufacturer, with different primers designed within the known heterologous insert DNA sequence (Round 2 "gene specific primers" or "GSP2"). The following reagents were mixed in a PCR tube on ice: 1 pl of the appropriate diluted primary PCR
product, 1 gl 10 M AP2, 1 I 10 M GSP2, 1 I 10 mM dNTPs, 5 I 10X Advantage PCR Buffer, 1 pl of BD Advantage 2 Polymerase, and 40 I distilled water. PCR
was completed using the following program: five cycles at 94 C for 25 seconds and 72 C for four minutes, 20 cycles at 94 C for 25 seconds and 67 C for four minutes, and one cycle at 67 C for four minutes. The reactions that worked were (1) the Drai and the Xmnl libraries with the GSP2 primer 162GW3F2:
5'AAGATTGAATCCTGTTGCCGGTCTTGCG3' (SEQ ID NO: 57) and primer AP2.
[00129] Ten I of the PCR products were run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli, and the plasmid DNA was extracted from the cells after overnight growth with a Qiagen Miniprep kit according to the manufacturer's instructions. This plasmid was sequenced using end run sequencing.
1001301 A new primer was designed within the new, previously unknown sequence to be used with a primer in the insert DNA to amplify approximately I kb of 3' flanking sequence out of the genomic DNA. The insert DNA primer 1620W3F1:
5TCTC1TGCTAAGCTGGGAGCTCGATCCG3' (SEQ ID NO: 56) and a 3' flanking sequence primer 1623'GWRI: 5CTGGTGAACCGA Full ACGGAGG3' (SEQ ID NO:
58) were used to amplify a nucleic acid molecule comprising the 3' flanking sequence for confirmation. The sequence of the resulting amplicon is set forth in SEQ ID
NO: 59. This 3' amplicon comprises the 3' junction sequence set forth in SEQ ID NO: 47. Ten p.1 of the PCR amplicon was run on a 1% agarose gel containing ethidium bromide. The appropriate band was extracted from the agarose gel and purified using a Qiagen Qiaquick Gel Extraction Kit according to the manufacturer's instructions. The extracted DNA was cloned into an Invitrogen TOPO-XL cloning vector according to the manufacturer's instructions. This clone was transformed into E. coli. Plasmid DNA was extracted from the cells after overnight growth in media with a Qiagen Miniprep kit according to the manufacturer's instructions. Three plasmids were completely sequenced using the primers shown in Table 5. The plasmid sequences were aligned to generate the complete confirmed 3' flanking sequence (SEQ ID NO: 48).
Table 5. Primer sequences.
Primer Name Sequence (5'33') Sequence No.
b00106a GATTGAATCCTGTTGCC SEQ ID NO: 81 b00106b TCTCATAAATAACGTCATGC SEQ ID NO: 82 b00108a TCTGTGGATAACCGTAITAC SEQ ED NO: 83 b00201h TTCACGGGAGACTTTATCTG SEQ ID NO: 60 b00605a CCGATTCATTAATGCAG SEQ ID NO: 61 b00704h AATAATATCACTCTGTACATCC SEQ ID NO: 64 b00712e AGTAACATAGATGACACCGC SEQ ID NO: 84 b011 06a CCAGTGTGCTGGAATTCG SEQ ID NO: 85 b01 106f GTTGTAAAACGACGG SEQ ID NO: 65 b011 07h CCAGTGTGATGGATATCTGC SEQ ID NO: 86 b01 108e CCAGTGTGCTGGAATTCG SEQ ID NO: 87 b01111f CCAGTGTGATGGATATCTGC SEQ ID NO: 88 b01709f TAGGCACCCCAGGCTTTA SEQ ID NO: 66 b02701a GTGTGCTGGAATTCGCCCTT SEQ ID NO: 89 b02701e TATCTGCAGAAITCGCCCTT SEQ ID NO: 90 b02702a GTGTGCTGGAA'FTCGCCCTT SEQ ID NO: 91 b02702e TATCTGCAGAATTCGCCCTT SEQ ID NO: 92 b02703a GTGTGCTGGAATTCGCCCTT SEQ ID NO: 93 b02703e TATCTGCAGAATTCGCCCTT SEQ ID NO: 94 b02704a GTGTGCTGGAATTCGCCCTT SEQ ID NO: 95 b02811a GGTCTTGCGATGATTATC SEQ ID NO: 96 b05104c GAGAGGAATGGCAGCAGA SEQ ID NO: 97 b05104d CATGACGGGTTTGAGATT SEQ ID NO: 98 b05104e AATCTCAAACCCGTCATO SEQ ID NO: 99 b05104f TCTGCTGCCATTCCTCTC SEQ ID NO: 100 b05104g GATCAACCCGGAGAGGAAT SEQ ID NO: 101 b05104h CCATGACGGGTTTGAGAT SEQ ID NO: 102 b05105c CAACCGACCTGACAAGTGAC SEQ ID NO: 103 b05 105e ATCTCAAACCCGTCATGG SEQ ID NO: 104 b05105f ATTCCTCTCCGGGITGATC SEQ ID NO: 105 Example 6. Detection of Vip3Aa20 Protein in MIR162 by ELISA
[00131] Extracts were prepared from MIR162 leaves, roots, pith, kernels, silk, pollen and whole plants. They were quantitatively analyzed for Vip3Aa20 by ELISA using immunoaffinity purified goat anti-Vip3A and Protein A-purified rabbit anti-Vip3A
polyclonal antibodies using art recognized ELISA procedures. Vip3Aa20 was detected in all tissues analyzed across all growth stages. The mean level of Vip3Aa20 protein detected in the whole plant at anthesis and seed maturity was 10 Rig fresh weight and 16 gig fresh weight, respectively. The mean level of Vip3Aa20 protein in leaves at anthesis was 22 gig fresh weight.
Example 7. Field Efficacy of MIR162.
[00132] The M1R162 event was tested in the field for efficacy against fall armyvvorm (FAW, Spodoptera frugiperda), corn earworm (CEW, Helicoverpa zea), black cutworm (BCW, Agrotis ipsilon), and European corn borer (ECB, Ostrinia nubilalis).
Performance of the MIR162 event was compared with that of Warrior (Syngenta, Inc.), a conventional insecticide standard applied at a rate of 11.2 g a.i./acre, the transgenic corn event Btll, comprising a crylAb gene, and a Btll X MIR162 hybrid, produced by crossing a Btll inbred line with a MIRI62 inbred line.
[00133] Twenty-eight trials were planted in 13 states that represented the major corn growing regions of the continental United States. Trials were planted in a randomized complete block design with four replicated plots per block. Plots were 17.5 row feet per treatment per replication. Planting density was targeted at approximately 30,000 plants/acre. Immuno-diagnostic strips were used to confirm the presence or absence of the Vip3Aa20 and Cryl Ab proteins in the different treatment groups.
[00134] Natural pest infestations were utilized in trials where populations were sufficiently high; where they were not, artificial infestations were carried out.
Artificial infestation with two 21d- to 3- instar larvae at VI-V2 was utilized in the BCW trials.
Plots were rated at 3, 7, and 14 days post-infestation. BCW damage was recorded as partially damaged plants and fully cut plants. FAW plots were rated 7 and 14 days post-infestation or after 3"1- instar larvae were observed in control plants. The following scale was used to evaluate PAW and CEW leaf damage:
0.01 ¨ No visible leaf damage I ¨ Pin-hole damage on a few leaves 2 ¨ Small amount of shot-hole damage on a few leaves 3 ¨ Shot-hole damage on several leaves 4¨ Shot-hole damage and lesions on a few leaves ¨ Lesions on several leaves 6 ¨ Large lesions on several leaves 7 ¨ Large lesions and portions eaten away on a few leaves 8 ¨ Large lesions and portions eaten away on several leaves 9 ¨ Large lesions and portions eaten away on most leaves [00135] Plant damage was assessed for both first and second generation ECB.
The following scale was used for rating first generation damage, typically when larvae were in the 3rd to 4th instar:
1 - No visible leaf damage 2 - Small amount of shot-hole injury on a few leaves 3 - Shot-hole injury common on several leaves 4 - Several leaves with shot-holes and elongated lesions 5 - Several leaves with elongated lesions 6 - Several leaves with elongated lesions about 2.5 cm 7 - Long lesions common on about one half of the leaves 8 - Long lesions common on about two-thirds of the leaves 9 - Most leaves with long lesions [00136] Second generation ECB damage was assessed three to four weeks after artificial infestation or the end of the peak egg laying period. The following measurements were taken: number live larvae/stalk, number live larvae/shank, number live larvae/ear, number of tunnels/stalk, cumulative tunnel length (cm)/stalk, cumulative tunnel length (cm)/shank, number tunnels/ear, cumulative tunnel length kernel damage (cm)/ear, and %
infested plants.
[00137] CEW trials were generally planted late to increase natural infestation levels.
Feeding damage to ears was evaluated when CEW larvae on control plants were at the =

=
L5-L6 growth stage. Ear ratings included recording the number of larvae observed per ear and length of visible kernel feeding measured from the ear tip to the average lowest kernel destroyed.
[001381 Results of the BCW field trial are shown in Table 6. Less than 3% of the MIR162 plants and Btll X MIR162 plants were cut by BCW larvae. Significant numbers of Btll and control plants were cut. Plants comprising the MIR162 genotype had less BCW
feeding damage than the conventional insecticide treated plants.
Table 6. Stalk damage ratings from five trials with BCW at 21 days after infestation. Damage was measured as percent of total plants cut.
Treatment % Cut Plants Btll 42 Btl 1 X MIR162 3 Warrior Insecticide 12 Negative Control 40 [00139] The FAW field trial results are shown in Table 7. FAW feeding damage was measured on a scale of 0.01 to 9. Mean feeding damage in the MIR] 62 hybrids was very low (< 1) and significantly lower than average damage observed in the Bill and conventional insecticide treatments. Insect pressure in these trials was heavy with approximately 50 to 100 neonate larvae/plant. Btl I provided some protection from damage, whereas the conventional insecticide treatment provided no protection, sustaining the same amount of damage as the control.
Table 7. Leaf feeding damage ratings from five trials for FAW. Mean damage ratings at 14 days after infestation are presented for each treatment.
Treatment Mean Leaf Damage Rating (0.01-9) MIR162 0.90 Bt11 2.52 Btl 1 X MIR162 0.84 Warrior Insecticide 3.60 =
Negative Control 3.78 [00140] Results of the trials to assess first generation ECB damage are presented in Table 8. ECB feeding damage was rated on a scale of 1-9. In these trials, MIR162 conferred minimal protection against ECB feeding damage. Btl 1 fully protected the plants from ECB feeding damage. The Btl 1 X MIR162 plants had the same level of protection as the Btl 1 plants. The conventional insecticide treatment provided better protection than the MIR162 trait but significantly less protection than that provided by Btl 1.
Table 8. Leaf feeding damage field trial. Mean damage ratings at 14 days after infestation.
Treatment Mean Leaf Damage Rating (1-9) MIR162 2.95 Btll 1.00 Bt11 X MIR162 1.00 Warrior Insecticide 2.05 Negative Control 3.88 [00141] Second generation ECB damage results are presented in Table 9. Feeding damaged was measured as cumulative tunnel length in each corn stalk (if more than one tunnel was found, tunnel lengths were summed). The Btl 1 and Btl 1 x MIR162 treatments provided strong protection against stalk boring, whereas no protection against tunneling was provided by MIR162 alone or the insecticide treatment.
Table 9. Stalk damage ratings from seven trials for second generation ECB
larvae measured in tunnel length (cm) per stalk. Measurements were taken three to four weeks after artificial infestation.
Treatment Mean Tunnel Length (cm) MIR162 5.46 Btll 0.37 Btll X MIR162 0.48 Warrior Insecticide 5.06 Negative Control 5.04 =
[00142] Results of trials to assess CEW damage are presented in Table 10.
Feeding damage was rated as length of kernel damage per ear, measured from the ear tip to the average lowest kernel destroyed. Significant ear damage was observed in the Btl 1, insecticide, and check plots. Btll provided some level of protection compared to untreated check and was comparable to the protection provided by the conventional insecticide treatment. MIR162 and Bt II X M1R162 provided almost complete protection = of the ears from CEW larval feeding damage.
Table 10. Ear damage. ratings from six trials for CEW measured as average length of feeding damage. Measurements were taken when CEW larvae were L5-L6 in check plants.
Treatment Mean Ear Damage (cm) MIR162 0.17 Bt11 2.24 Btll X MIR162 0.02 Warrior Insecticide 2.20 Negative Control 3.42 Example 8. Efficacy of MIR162 Against Western Bean Cutworm.
[00143] Current commercial transgenic events producing Cry I Ab protein have not provided acceptable levels of protection against the western bean cutworm (WBCW, Striacosta albicosta). Therefore, MIR 162 alone and stacked with other transgenic = genotypes was tested for efficacy against WBCW.
[00144] WBCW eggs were collected from wild caught female moths. Larvae were fed on a rneridic black cutworm diet until use in the experiments. Corn plants were field grown.
The following treatments were tested: M1R162, Btl 1, MIR604, MIR162 X Btl 1, MIR162 =
X MIR604, MIR604 X Btl I, Forces (Syngenta, Inc.), a conventional insecticide applied at planting to a negative isoline, and two negative control isolines. MIR. 604 is a novel transgenic corn event that comprises a ay3A0.55 gene encoding a protein that is active against corn rootworm (Diabrotica spp.) larvae and is disclosed in US Patent Application 'publication No. 2005/0216970, published September 29, 2005.
= =
= 4 7 =
[00145] For the experiments, a two-inch piece of green silks and husk was cut from ears from field grown corn plants in each treatment and replication. The terminal brown ends of the silks were removed and the husk discarded. Approximately 1.5 inches of silks were placed in individual 14 ml plastic cups. One larva was then placed in each cup and the cups sealed. Several different stages of larvae were tested ranging from 3rd to 6th-instars.
Cups containing silks and larvae were held at natural day length and room temperature for the duration of the experiments. Larval survival was recorded after eight days. Treatments were replicated four times per experiment.
[00146] Results of the WBCW experiments are presented in Table 11. Survival of WBCW
on silks from the negative isolines and the conventional insecticide treatment were nearly 100%. Survival of WBCW larvae on Btll and MIR604 silks, tested either alone or in combination in the same plant, was not different from survival on the negative isolines.
Survival of WBCW larvae was reduced when larvae were fed silks from MIR162.
The combination of MIR162 X Btl 1 in the same plant did not decrease survival any further than MIR162 alone. However, surprisingly, when the MIR162 transgenic genotype was stacked with the MIR604 transgenic genotype in the same plant, larval mortality significantly increased compared to MIR162 or MIR604 alone.
Table 11. Percent ( SE) survival of WBCW larvae on corn silks.
Experiment Number Treatment 1 2 3 4 5 6 7 BO 1 75(25) 75(25) 100(0) 100(0) 100(0) 100(0) 100(0) MIR162 25(25) 0(0) 0(0) 50(29) 50(29) 50(29) 0(0) M1R604 100(0) 100(0) 100(0) MIR162xBtll 25(25) 25(25) 0(0) 0(0) 25(25) 25(25) 25(25) MIR162xMIR604 0(0) 25(25) 50(29) MIR604xBt11 75(25) Force 100(0) 100(0) 100(0) Neg. Control #1 100(0) 75(25) 100(0) 100(0) 75(25) 100(0) 100(0) Neg. Control #2 100(0) 100(0) 100(0) 100(0) 100(0) 100(0) 100(0) Example 9. Use of event MIR162 insertion site for targeted integration in maize.
[001471 The MIR162 flanking sequences disclosed in SEQ ID NO: 46 and SEQ ID
NO: 48 were used to search maize genomic databases. Identical matches to both flanking sequences where found on a BAC clone, CI-1201-307P5, of chromosome 5 (NCB!
Accession No. AC185313) on Contig 13 (SEQ ID NO: 106). More specifically, the MIR162 insert is on chromosome 5 between a 5' molecular marker, designated herein as the Opie2 marker (nucleotides 1680-3338 of SEQ ID NO: 106), and a 3' molecular marker, designated herein as the gag marker (nucleotides 43,27545,086 of SEQ
ID NO:
106). Using this information, it was determined that the heterologous DNA
inserted into MIR162 displaced 58 nucleotides of maize genomic DNA, nucleotides 25,455 to 25,512 of SEQ ID NO: 106 (also shown as SEQ ID NO: 107), which is between the 5' flanking sequence (nucleotides 1-25,454 of SEQ ID NO: 106) and the 3' flanking sequence (nucleotides 25,513-51,328 of SEQ ID NO: 106).
[00148] Consistent agronomic performance of the transgene of event MIR162 over several generations under field conditions suggests that these identified regions around the MIRI62 insertion site provide good genomic locations for the targeted integration of other transgenic genes of interest. Such targeted integration overcomes the problems with so-called "positions effects," and the risk of creating a mutation in the genome upon integration of the transgene into the host. Further=advantages of such targeted integration include, but are not limited to, reducing the large number of transformation events that must be screened and tested before obtaining a transgenic plant that exhibits the desired level of transgene expression without also exhibiting abnormalities resulting from the inadvertent insertion of the transgene into an important locus in the host genome.
Moreover, such targeted integration allows for stacking transgenes rendering the breeding of elite plant lines with both genes more efficient.
[001491 Using the above disclosed teaching, the skilled person is able to use methods know in the art to target heterologous nucleic acids of interest to the same insertion site on chromosome 5 as that in MIRI62 or to a site in close proximity to the insertion site in MIR162. One such method, is disclosed in US Patent Application Publication No.

20060253918. Briefly, up to 20 Kb of the = genomic sequence flanking 5' to the insertion site (nucleotides 5,454 to 25,454 of SEQ ID -NO: 106) and up to 20 Kb of the genomic sequence flanking 3' to the insertion site (nucleotides 25,513 to 45,513 of SEQ OD NO: 106) are used to flank the gene or genes of interest that are intended to be inserted into a genomic location on Chromosome 5 via homologous recombination. These sequences can be further flanked by T-DNA
border repeats such as the left border (LB) and right border (RB) repeat sequences and other booster sequences for enhancing T-DNA delivery efficiency. The gene or genes of interest can be placed exactly as in the MIRI 62 insertion site or can be placed anywhere within the 20 Kb regions around the MIRI62 insertion sites to confer consistent level of transgene expression without detrimental effects on the plant. The DNA vectors containing the gene or genes of interest and flanking sequences can be delivered into plant cells via one of the several methods known to those skilled in the art, including but not limited to Agrobacterium-mediated transformation. The insertion of the DNA
vector into the MIR162 target site can be further enhanced by one of the several methods, including but not limited to the co-expression or up-regulation of recombination enhancing genes or down-regulation of endogenous recombination suppression genes.
Furthermore, it is known in the art that cleavage of specific sequences in the genome can be used to increase homologous recombination frequency, therefore insertion into the MIR162 insertion site and its flanking regions can be enhanced by expression of natural or designed sequence-specific endonucleases for cleaving these sequences.
Thus, using the teaching provided herein, any heterologous nucleic acid can be inserted on maize chromosome 5 at a target site located between nucleotides 25,454 and 45,513 of SEQ ID
NO: 106 or a target site in the vicinity to this site.
Example 10. Use of event MIR162 insertion site and flanking sequences for stabilization of gene expression.
[001501 The genomic sequences flanking the MIR162 insertion site may also be used to stabilize expression of other gene(s) of interest when inserted as a transgene in other genomic locations in maize and other crops. Specifically, up to 20 Kb of the genomic sequence flanking 5' to the insertion site (nucleotides 5,454 to 25,454 of SEQ
ID NO:
106) and up to 20 Kb of the genomic sequence flanking 3' to the insertion site (nucleotides 25,513 to 45,513 of SEQ OD NO: 106) are used to flank the gene or genes of interest that are intended to be inserted into the genome of plants. These sequences can be further flanked by T-DNA border repeats such as the left border (LB) and right border (RB) repeat sequences and other booster sequences for enhancing 1-DNA delivery efficiency. The gene or genes of interest can be placed exactly as in the MIR162 insertion =
site or can be placed anywhere within the 20 Kb regions around the MIR162 insertion sites to confer consistent level of transgene expression. The DNA vectors containing the gene or genes of interest and MIR162 insertion site flanking sequence can be delivered into plant cells via one of the several methods known to those skilled in the art, including but not limited to protoplast transformation, biolistic bombardment and Agrobacterium-.
mediated transformation. The delivered DNA can be integrated randomly into a plant genome or can also be present as part of the independently segregating genetic units such as artificial chromosome or mini-chromosome. The DNA vectors containing the gene(s) of interest and the MIR162 insertion site flanking sequences can be delivered into plant cells. Thus, by surrounding a gene or genes of interest with the genomic sequence flanking the MIR162 insertion site, the expression of such genes are stabilized in a transgenic host plant such as a dicot plant or a monocot plant like corn.
DEPOSIT
=
[00151] Applicants have made a deposit of corn seed of event MIR162 disclosed above on 23 January 2007 in accordance with the Budapest Treaty at the American Type Culture Collection (ATCC), 1801 University Boulevard, Manassas, VA 20110 under ATCC
Accession No. PTA-8166. The deposit will be maintained in the depositary fora period of 30 years, or 5 years after the last request,. or the effective life of the patent, whichever is longer, and will be replaced as necessary during that period. Applicants impose no restrictions on the availability of the deposited material from the ATCC;
however, Applicants have no authority to waive any restrictions imposed by law on the transfer of biological material or its transportation in commerce. Applicants do not waive any infringement of their rights granted under this patent or under the Plant Variety Protection Act (7 USC 2321 et seq.).

SEQUENCE LISTING IN ELECTRONIC FORM
In accordance with Section 111(1) of the Patent Rules, this description contains a sequence listing in electronic form in ASCII text format (file: 30506-87D Seq 19-NOV-08 vl.txt).
A copy of the sequence listing in electronic form is available from the Canadian Intellectual Property Office.
The sequences in the sequence listing in electronic form are reproduced in the following table.
SEQUENCE TABLE
<110> Syngenta Participations AG
Long, NyKoll Pulliam, Derrick Hart, Hope Bottoms, Jeff Meghji, Moez Que, Qiudeng <120> Corn Event MIR162 <130> 30506-87D
<160> 107 <170> PatentIn version 3.3 <210> 1 <211> 2370 <212> DNA
<213> Artificial Sequence <220>
<223> vip3Aa20 coding sequence <400> 1 atgaacaaga acaacaccaa gctgagcacc cgcgccctgc cgagcttcat cgactacttc 60 aacggcatct acggcttcgc caccggcatc aaggacatca tgaacatgat cttcaagacc 120 gacaccggcg gcgacctgac cctggacgag atcctgaaga accagcagct gctgaacgac 180 atcagcggca agctggacgg cgtgaacggc agcctgaacg acctgatcgc ccagggcaac 240 ctgaacaccg agctgagcaa ggagatcctt aagatcgcca acgagcagaa ccaggtgctg 300 aacgacgtga acaacaagct ggacgccatc aacaccatgc tgcgcgtgta cctgccgaag 360 atcaccagca tgctgagcga cgtgattaag cagaactacg ccctgagcct gcagatcgag 420 tacctgagca agcagctgca ggagatcagc gacaagctgg acatcatcaa cgtgaacgtc 480 ctgatcaaca gcaccctgac cgagatcacc ccggcctacc agcgcatcaa gtacgtgaac 540 gagaagttcg aagagctgac cttcgccacc gagaccagca gcaaggtgaa gaaggacggc 600 agcccggccg acatcctgga cgagctgacc gagctgaccg agctggcgaa gagcgtgacc 660 aagaacgacg tggacggctt cgagttctac ctgaacacct tccacgacgt gatggtgggc 720 aacaacctgt tcggccgcag cgccctgaag accgccagcg agctgatcac caaggagaac 780 gtgaagacca gcggcagcga ggtgggcaac gtgtacaact tcctgatcgt gctgaccgcc 840 ctgcaggccc aggccttcct gaccctgacc acctgtcgca agctgctggg cctggccgac 900 51a atcgactaca ccagcatcat gaacgagcac ttgaacaagg agaaggagga gttccgcgtg 960 aacatcctgc cgaccctgag caacaccttc agcaacccga actacgccaa ggtgaagggc 1020 agcgacgagg acgccaagat gatcgtggag gctaagccgg gccacgcgtt gatcggcttc 1080 gagatcagca acgacagcat caccgtgctg aaggtgtacg aggccaagct gaagcagaaC 1140 taccaggtgg acaaggacag cttgagcgag gtgatctacg gcgacatgga caagctgctg 1200 tgtccggacc agagcgagca aatctactac accaacaaca tcgtgttccc gaacgagtac 1260 gtgatcacca agatcgactt caccaagaag atgaagaccc tgcgctacga ggtgaccgcc 1320 aacttctacg acagcagcac cggcgagatc gacctgaaca agaagaaggt ggagagcagc 1380 gaggccgagt accgcaccct gagcgcgaac gacgacggcg tctacatgcc actgggcgtg 1440 atcagcgaga ccttcctgac cccgatcaac ggctttggcc tgcaggccga cgagaacagc 1500 cgcctgatca ccctgacctg taagagctac ctgcgcgagc tgctgctagc caccgacctg 1560 agcaacaagg agaccaagct gatcgtgcca ccgagcggct tcatcagcaa catcgtggag 1620 aacggcagca tcgaggagga caacctggag ccgtggaagg ccaacaacaa gaacgcctac 1680 gtcgaccaca ccggcggcgt gaacggcacc aaggccctgt acgtgcacaa ggacggcggc 1740 atcagccagt tcatcggcga caagctgaag ccgaagaccg agtacgtgat ccagtacacc 1800 gtgaagggca agccatcgat tcacctgaag gacgagaaca ccggctacat ccactacgag 1860 gacaccaaca acaacctgga ggactaccag accatcaaca agcgcttcac caccggcacc 1920 gacctgaagg gcgtgtacct gatcctgaag agccagaacg gcgacgaggc ctggggcgac 1980 aacttcatca tcctggagat cagcccgagc gagaagctgc tgagcccgga gctgatcaac 2040 accaacaact ggaccagcac cggcagcacc aacatcagcg gcaacaccct gaccctgtac 2100 cagggcggcc gcggcatcct gaagcagaac ctgcagctgg acagcttcag cacctaccgc 2160 gtgtacttca gcgtgagcgg cgacgccaac gtgcgcatcc gcaactcccg cgaggtgctg 2220 ttcgagaaga ggtacatgag cggcgccaag gacgtgagcg agatgttcac caccaagttc 2280 gagaaggaca acttctacat cgagctgagc cagggcaaca acctgtacgg cggcccgatc 2340 gtgcacttct acgacgtgag catcaagtag 2370 <210> 2 <211> 789 <212> PRT
<213> Artificial Sequence <220>
<223> Vip3Aa toxin <220>
<221> MISC FEATURE
<222> (1)..(789) <223> Vip3Aa20 protein produced by MIR162 <400> 2 Met Asn Lys Asn Asn Thr Lys Leu Ser Thr Arg Ala Leu Pro Ser Phe Ile Asp Tyr Phe Asn Gly Ile Tyr Gly Phe Ala Thr Gly Ile Lys Asp Ile Met Asn Met Ile Phe Lys Thr Asp Thr Gly Gly Asp Leu Thr Leu Asp Glu Ile Leu Lys Asn Gln Gln Leu Leu Asn Asp Ile Ser Gly Lys Leu Asp Gly Val Asn Gly Ser Leu Asn Asp Leu Ile Ala Gln Gly Asn Leu Asn Thr Glu Leu Ser Lys Glu Ile Leu Lys Ile Ala Asn Glu Gln Asn Gln Val Leu Asn Asp Val Asn Asn Lys Leu Asp Ala Ile Asn Thr Met Leu Arg Val Tyr Leu Pro Lys Ile Thr Ser Met Leu Ser Asp Val 51b Ile Lys Gln Asn Tyr Ala Leu Ser Leu Gln Ile Glu Tyr Leu Ser Lys Gln Leu Gln Glu Ile Ser Asp Lys Leu Asp Ile Ile Asn Val Asn Val Leu Ile Asn Ser Thr Leu Thr Glu Ile Thr Pro Ala Tyr Gln Arg Ile Lys Tyr Val Asn Glu Lys Phe Glu Glu Leu Thr Phe Ala Thr Glu Thr Ser Ser Lys Val Lys Lys Asp Gly Ser Pro Ala Asp Ile Leu Asp Glu Leu Thr Glu Leu Thr Glu Leu Ala Lys Ser Val Thr Lys Asn Asp Val Asp Gly Phe Glu Phe Tyr Leu Asn Thr Phe His Asp Val Met Val Gly Asn Asn Leu Phe Gly Arg Ser Ala Leu Lys Thr Ala Ser Glu Leu Ile Thr Lys Glu Asn Val Lys Thr Ser Gly Ser Glu Val Gly Asn Val Tyr Asn Phe Leu Ile Val Leu Thr Ala Leu Gln Ala Gln Ala Phe Leu Thr Leu Thr Thr Cys Arg Lys Leu Leu Gly Leu Ala Asp Ile Asp Tyr Thr Ser Ile Met Asn Glu His Leu Asn Lys Glu Lys Glu Glu Phe Arg Val Asn Ile Leu Pro Thr Leu Ser Asn Thr Phe Ser Asn Pro Asn Tyr Ala Lys Val Lys Gly Ser Asp Glu Asp Ala Lys Met Ile Val Glu Ala Lys Pro Gly His Ala Leu Ile Gly Phe Glu Ile Ser Asn Asp Ser Ile Thr Val Leu Lys Val Tyr Glu Ala Lys Leu Lys Gln Asn Tyr Gln Val Asp Lys Asp Ser Leu Ser Glu Val Ile Tyr Gly Asp Met Asp Lys Leu Leu Cys Pro Asp Gln Ser Glu Gln Ile Tyr Tyr Thr Asn Asn Ile Val Phe Pro Asn Glu Tyr Val Ile Thr Lys Ile Asp Phe Thr Lys Lys Met Lys Thr Leu Arg Tyr Glu Val Thr Ala Asn Phe Tyr Asp Ser Ser Thr Gly Glu Ile Asp Leu Asn Lys Lys Lys Val Glu Ser Ser Glu Ala Glu Tyr Arg Thr Leu Ser Ala Asn Asp Asp Gly Val Tyr Met Pro Leu Gly Val Ile Ser Glu Thr Phe Leu Thr Pro Ile Asn Gly Phe Gly Leu Gln Ala Asp Glu Asn Ser Arg Leu Ile Thr Leu Thr Cys Lys Ser Tyr Leu Arg Glu Leu Leu Leu Ala Thr Asp Leu Ser Asn Lys Glu Thr Lys Leu Ile Val Pro Pro Ser Gly Phe Ile Ser Asn Ile Val Glu Asn Gly Ser Ile Glu Glu Asp Asn Leu Glu Pro Trp Lys Ala Asn Asn Lys Asn Ala Tyr Val Asp His Thr Gly Gly Val Asn Gly Thr Lys Ala Leu Tyr Val His Lys Asp Gly Gly Ile Ser Gln Phe Ile Gly Asp Lys Leu Lys Pro Lys 51c Thr Glu Tyr Val Ile Gin Tyr Thr Val Lys Gly Lys Pro Ser Ile His Leu Lys Asp Glu Asn Thr Gly Tyr Ile His Tyr Glu Asp Thr Asn Asn Asn Leu Glu Asp Tyr Gin Thr Ile Asn Lys Arg Phe Thr Thr Gly Thr Asp Leu Lys Gly Val Tyr Leu Ile Leu Lys Ser Gin Asn Gly Asp Glu Ala Trp Gly Asp Asn Phe Ile Ile Leu Glu Ile Ser Pro Ser Glu Lys Leu Leu Ser Pro Glu Leu Ile Asn Thr Asn Asn Trp Thr Ser Thr Gly Ser Thr Asn Ile Ser Gly Asn Thr Leu Thr Leu Tyr Gin Gly Gly Arg Gly Ile Leu Lys Gin Asn Leu Gin Leu Asp Ser Phe Ser Thr Tyr Arg Val Tyr Phe Ser Val Ser Gly Asp Ala Asn Val Arg Ile Arg Asn Ser Arg Glu Val Leu Phe Glu Lys Arg Tyr Met Ser Gly Ala Lys Asp Val Ser Glu Met Phe Thr Thr Lys Phe Glu Lys Asp Asn Phe Tyr Ile Glu Leu Ser Gin Gly Asn Asn Leu Tyr Gly Gly Pro Ile Val His Phe Tyr Asp Val Ser Ile Lys <210> 3 <211> 14405 <212> DNA
<213> Artificial Sequence <220>
<223> Plasmid pNOV1300 <400> 3 atgaacaaga acaacaccaa gctgagcacc cgcgccctgc cgagcttcat cgactacttc 60 aacggcatct acggcttcgc caccggcatc aaggacatca tgaacatgat cttcaagacc 120 gacaccggcg gcgacctgac cctggacgag atcctgaaga accagcagct gctgaacgac 180 atcagcggca agctggacgg cgtgaacggc agcctgaacg acctgatcgc ccagggcaac 240 ctgaacaccg agctgagcaa ggagatcctt aagatcgcca acgagcagaa ccaggtgctg 300 aacgacgtga acaacaagct ggacgccatc aacaCcatgc tgcgcgtgta cctgccgaag 360 atcaccagca tgctgagcga cgtgatgaag cagaactacg ccctgagcct gcagatcgag 420 tacctgagca agcagctgca ggagatcagc gacaagctgg acatcatcaa cgtgaacgtc 480 ctgatcaaca gcaccctgac cgagatcacc ccggcctacc agcgcatcaa gtacgtgaac 540 gagaagttcg aagagctgac cttcgccacc gagaccagca gcaaggtgaa gaaggacggc 600 agcccggccg acatcctgga cgagctgacc gagctgaccg agctggcgaa gagcgtgacc 660 aagaacgacg tggacggctt cgagttctac ctgaacacct tccacgacgt gatggtgggc 720 aacaacctgt tcggccgcag cgccctgaag accgccagcg agctgatcac caaggagaac 780 gtgaagacca gcggcagcga ggtgggcaac gtgtacaact tcctgatcgt gctgaccgcc 840 ctgcaggccc aggccttcct gaccctgacc acctgtcgca agctgctggg cctggccgac 900 atcgactaca ccagcatcat gaacgagcac ttgaacaagg agaaggagga gttccgcgtg 960 aacatcctgc cgaccctgag caacaccttc agcaacccga actacgccaa ggtgaagggc 1020 agcgacgagg acgccaagat gatcgtggag gctaagccgg gccacgcgtt gatcggcttc 1080 gagatcagca acgacagcat caccgtgctg aaggtgtacg aggccaagct gaagcagaac 1140 taccaggtgg acaaggacag cttgagcgag gtgatctacg gcgacatgga caagctgctg 1200 tgtccggacc agagcgagca aatctactac accaacaaca tcgtgttccc gaacgagtac 1260 51d gtgatcacca agatcgactt caccaagaag atgaagaccc tgcgctacga ggtgaccgcc 1320 aacttctacg acagcagcac cggcgagatc gacctgaaca agaagaaggt ggagagcagc 1380 gaggccgagt accgcaccct gagcgcgaac gacgacggcg tctacatgcc actgggcgtg 1440 atcagcgaga ccttcctgac cccgatcaac ggctttggcc tgcaggccga cgagaacagc 1500 cgcctgatca ccctgacctg taagagctac ctgcgcgagc tgctgctagc caccgacctg 1560 agcaacaagg agaccaagct gatcgtgcca ccgagcggct tcatcagcaa catcgtggag 1620 aacggcagca tcgaggagga caacctggag ccgtggaagg ccaacaacaa gaacgcctac 1680 gtggaccaca ccggcggcgt gaacggcacc aaggccctgt acgtgcacaa ggacggcggc 1740 atcagccagt tcatcggcga caagctgaag ccgaagaccg agtacgtgat ccagtacacc 1800 gtgaagggca agccatcgat tcacctgaag gacgagaaca ccggctacat ccactacgag 1860 gacaccaaca acaacctgga ggactaccag accatcaaca agcgcttcac caccggcacc 1920 gacctgaagg gcgtgtacct gatcctgaag agccagaacg gcgacgaggc ctggggcgac 1980 aacttcatca tcctggagat cagcccgagc gagaagctgc tgagcccgga gctgatcaac 2040 accaacaact ggaccagcac cggcagcacc aacatcagcg gcaacaccct gaccctgtac 2100 cagggcggcc gcggcatcct gaagcagaac ctgcagctgg acagcttcag cacctaccgc 2160 gtgtacttca gcgtgagcgg cgacgccaac gtgcgcatcc gcaactcccg cgaggtgctg 2220 ttcgagaaga ggtacatgag cggcgccaag gacgtgagcg agatgttcac caccaagttc 2280 gagaaggaca acttctacat cgagctgagc cagggcaaca acctgtacgg cggcccgatc 2340 gtgcacttct acgacgtgag catcaagtag gagctctaga tctgttctgc acaaagtgga 2400 gtagtcagtc atcgatcagg aaccagacac cagactttta ttcatacagt gaagtgaagt 2460 gaagtgcagt gcagtgagtt gctggttttt gtacaactta gtatgtattt gtatttgtaa 2520 aatacttcta tcaataaaat ttctaattcc taaaaccaaa atccaggggt accagcttgc 2580 atgcctgcag tgcagcgtga cccggtcgtg cccctctcta gagataatga gcattgcatg 2640 tctaagttat aaaaaattac cacatatttt ttttgtcaca cttgtttgaa gtgcagttta 2700 tctatcttta tacatatatt taaactttac tctacgaata atataatcta tagtactaca 2760 ataatatcag tgttttagag aatcatataa atgaacagtt agacatggtc taaaggacaa 2820 ttgagtattt tgacaacagg actctacagt tttatctttt tagtgtgcat gtgttctcct 2880 ttttttttgc aaatagcttc acctatataa tacttcatcc attttattag tacatccatt 2940 tagggtttag ggttaatggt ttttatagac taattttttt agtacatcta ttttattcta 3000 ttttagcctc taaattaaga aaactaaaac tctattttag tttttttatt taataattta 3060 gatataaaat agaataaaat aaagtgacta aaaattaaac aaataccctt taagaaatta 3120 aaaaaactaa ggaaacattt ttcttgtttc gagtagataa tgccagcctg ttaaacgccg 3180 tcgacgagtc taacggacac caaccagcga accagcagcg tcgcgtcggg ccaagcgaag 3240 cagacggcac ggcatctctg tcgctgcctc tggacccctc tcgagagttc cgctccaccg 3300 ttggacttgc tccgctgtcg gcatccagaa attgcgtggc ggagcggcag acgtgagccg 3360 gcacggcagg cggcctcctc ctcctctcac ggcaccggca gctacggggg attcctttcc 3420 caccgctcct tcgctttccc ttcctcgccc gccgtaataa atagacaccc cctccacacc 3480 ctctttcccc aacctcgtgt tgttcggagc gcacacacac acaaccagat ctcccccaaa 3540 tccacccgtc ggcacctccg cttcaaggta cgccgctcgt cctccccccc cccccctctc 3600 taccttctct agatcggcgt tccggtccat ggttagggcc cggtagttct acttctgttc 3660 atgtttgtgt tagatccgtg tttgtgttag atccgtgctg ctagcgttcg tacacggatg 3720 cgacctgtac gtcagacacg ttctgattgc taacttgcca gtgtttctct ttggggaatc 3780 ctgggatggc tctagccgtt ccgcagacgg gatcgatttc atgatttttt ttgtttcgtt 3840 gcatagggtt tggtttgccc ttttccttta tttcaatata tgccgtgcac ttgtttgtcg 3900 ggtcatcttt tcatgctttt ttttgtcttg gttgtgatga tgtggtctgg ttgggcggtc 3960 gttctagatc ggagtagaat tctgtttcaa actacctggt ggatttatta attttggatc 4020 tgtatgtgtg tgccatacat attcatagtt acgaattgaa gatgatggat ggaaatatcg 4080 atctaggata ggtatacatg ttgatgcggg ttttactgat gcatatacag agatgctttt 4140 tgttcgcttg gttgtgatga tgtggtgtgg ttgggcggtc gttcattcgt tctagatcgg 4200 agtagaatac tgtttcaaac tacctggtgt atttattaat tttggaactg tatgtgtgtg 4260 tcatacatct tcatagttac gagtttaaga tggatggaaa tatcgatcta ggataggtat 4320 acatgttgat gtgggtttta ctgatgcata tacatgatgg catatgcagc atctattcat 4380 atgctctaac cttgagtacc tatctattat aataaacaag tatgttttat aattattttg 4440 atcttgatat acttggatga tggcatatgc agcagctata tgtggatttt tttagccctg 4500 ccttcatacg ctatttattt gcttggtact gtttcttttg tcgatgctca ccctgttgtt 4560 tggtgttact tctgcaggga tccccgatca tgcaaaaact cattaactca gtgcaaaact 4620 atgcctgggg cagcaaaacg gcgttgactg aactttatgg tatggaaaat ccgtccagcc 4680 agccgatggc cgagctgtgg atgggcgcac atccgaaaag cagttcacga gtgcagaatg 4740 51e ccgccggaga tatcgtttca ctgcgtgatg tgattgagag tgataaatcg actctgctcg 4800 gagaggccgt tgccaaacgc tttggcgaac tgcctttcct gttcaaagta ttatgcgcag 4860 cacagccact ctccattcag gttcatccaa acaaacacaa ttctgaaatc ggttttgcca 4920 aagaaaatgc cgcaggtatc ccgatggatg ccgccgagcg taactataaa gatcctaacc 4980 acaagccgga gctggttttt gcgctgacgc ctttccttgc gatgaacgcg tttcgtgaat 5040 tttccgagat tgtctcccta ctccagccgg tcgcaggtgc acatccggcg attgctcact 5100 ttttacaaca gcctgatgcc gaacgtttaa gcgaactgtt cgccagcctg ttgaatatgc 5160 agggtgaaga aaaatcccgc gcgctggcga ttttaaaatc ggccctcgat agccagcagg 5220 gtgaaccgtg gcaaacgatt cgtttaattt ctgaatttta cccggaagac agcggtctgt 5280 tctccccgct attgctgaat gtggtgaaat tgaaccctgg cgaagcgatg ttcctgttcg 5340 ctgaaacacc gcacgcttac ctgcaaggcg tggcgctgga agtgatggca aactccgata 5400 acgtgctgcg tgcgggtctg acgcctaaat acattgatat tccggaactg gttgccaatg 5460 tgaaattcga agccaaaccg gctaaccagt tgttgaccca gccggtgaaa caaggtgcag 5520 aactggactt cccgattcca gtggatgatt ttgccttctc gctgcatgac cttagtgata 5580 aagaaaccac cattagccag cagagtgccg ccattttgtt ctgcgtcgaa ggcgatgcaa 5640 cgttgtggaa aggttctcag cagttacagc ttaaaccggg tgaatcagcg tttattgccg 5700 ccaacgaatc accggtgact gtcaaaggcc acggccgttt agcgcgtgtt tacaacaagc 5760 tgtaagagct tactgaaaaa attaacatct cttgctaagc tgggagctcg atccgtcgac 5820 ctgcagatcg ttcaaacatt tggcaataaa gtttcttaag attgaatcct gttgccggtc 5880 ttgcgatgat tatcatataa tttctgttga attacgttaa gcatgtaata attaacatgt 5940 aatgcatgac gttatttatg agatgggttt ttatgattag agtcccgcaa ttatacattt 6000 aatacgcgat agaaaacaaa atatagcgcg caaactagga taaattatcg cgcgcggtgt 6060 catctatgtt actagatccc cgggtctaga caattcagta cattaaaaac gtccgcaatg 6120 tgttattaag ttgtctaagc gtcaatttgt ttacaccaca atatatcctg ccaccagcca 6180 gccaacagct ccccgaccgg cagctcggca caaaatcacc actcgataca ggcagcccat 6240 cagtccggga cggcgtcagc gggagagccg ttgtaaggcg gcagactttg ctcatgttac 6300 cgatgctatt cggaagaacg gcaactaagc tgccgggttt gaaacacgga tgatctcgcg 6360 gagggtagca tgttgattgt aacgatgaca gagcgttgct gcctgtgatc aaatatcatc 6420 tccctcgcag agatccgaat tatcagcctt cttattcatt tctcgcttaa ccgtgacagg 6480 ctgtcgatct tgagaactat gccgacataa taggaaatcg ctggataaag ccgctgagga 6540 agctgagtgg cgctatttct ttagaagtga acgttgacga tcgtcgaccg taccccgatg 6600 aattaattcg gacgtacgtt ctgaacacag ctggatactt acttgggcga ttgtcataca 6660 tgacatcaac aatgtacccg tttgtgtaac cgtctcttgg aggttcgtat gacactagtg 6720 gttcccctca gcttgcgact agatgttgag gcctaacatt ttattagaga gcaggctagt 6780 tgcttagata catgatcttc aggccgttat ctgtcagggc aagcgaaaat tggccattta 6840 tgacgaccaa tgccccgcag aagctcccat ctttgccgcc atagacgccg cgcccccctt 6900 ttggggtgta gaacatcctt ttgccagatg tggaaaagaa gttcgttgtc ccattgttgg 6960 caatgacgta gtagccggcg aaagtgcgag acccatttgc gctatatata agcctacgat 7020 ttccgttgcg actattgtcg taattggatg aactattatc gtagttgctc tcagagttgt 7080 cgtaatttga tggactattg tcgtaattgc ttatggagtt gtcgtagttg cttggagaaa 7140 tgtcgtagtt ggatggggag tagtcatagg gaagacgagc ttcatccact aaaacaattg 7200 gcaggtcagc aagtgcctgc cccgatgcca tcgcaagtac gaggcttaga accaccttca 7260 acagatcgcg catagtcttc cccagctctc taacgcttga gttaagccgc gccgcgaagc 7320 ggcgtcggct tgaacgaatt gttagacatt atttgccgac taccttggtg atctcgcctt 7380 tcacgtagtg aacaaattct tccaactgat ctgcgcgcga ggccaagcga tcttcttgtc 7440 caagataagc ctgcctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca 7500 ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa 7560 tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc 7620 atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga 7680 gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga 7740 tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct 7800 gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt 7860 gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag 7920 tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg 7980 taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca 8040 aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata 8100 gttgagtcga tacttcggcg atcaccgctt ccctcatgat gtttaactcc tgaattaagc 8160 cgcgccgcga agcggtgtcg gcttgaatga attgttaggc gtcatcctgt gctcccgaga 8220 51f accagtacca gtacatcgct gtttcgttcg agacttgagg tctagtttta tacgtgaaca 8280 ggtcaatgcc gccgagagta aagccacatt ttgcgtacaa attgcaggca ggtacattgt 8340 tcgtttgtgt ctctaatcgt atgccaagga gctgtctgct tagtgcccac tttttcgcaa 8400 attcgatgag actgtgcgcg actcctttgc ctcggtgcgt gtgcgacaca acaatgtgtt 8460 cgatagaggc tagatcgttc catgttgagt tgagttcaat cttcccgaca agctcttggt 8520 cgatgaatgc gccatagcaa gcagagtctt catcagagtc atcatccgag atgtaatcct 8580 tccggtaggg gctcacactt ctggtagata gttcaaagcc ttggtcggat aggtgcacat 8640 cgaacacttc acgaacaatg aaatggttct cagcatccaa tgtttccgcc acctgctcag 8700 ggatcaccga aatcttcata tgacgcctaa cgcctggcac agcggatcgc aaacctggcg 8760 cggcttttgg cacaaaaggc gtgacaggtt tgcgaatccg ttgctgccac ttgttaaccc 8820 ttttgccaga tttggtaact ataatttatg ttagaggcga agtcttgggt aaaaactggc 8880 ctaaaattgc tggggatttc aggaaagtaa acatcacctt ccggctcgat gtctattgta 8940 gatatatgta gtgtatctac ttgatcgggg gatctgctgc ctcgcgcgtt tcggtgatga 9000 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 9060 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggcgc 9120 agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta tgcggcatca 9180 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 9240 agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 9300 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 9360 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 9420 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 9480 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 9540 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 9600 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 9660 agttcggtgt agg'tcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 9720 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 9780 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 9840 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 9900 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 9960 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 10020 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 10080 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 10140 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 10200 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 10260 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 10320 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 10380 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 10440 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 10500 aacgttgttg ccattgctgc aggggggggg gggggggggt tccattgttc attccacgga 10560 caaaaacaga gaaaggaaac gacagaggcc aaaaagctcg ctttcagcac ctgtcgtttc 10620 ctttcttttc agagggtatt ttaaataaaa acattaagtt atgacgaaga agaacggaaa 10680 cgccttaaac cggaaaattt tcataaatag cgaaaacccg cgaggtcgcc gccccgtaac 10740 ctgtcggatc accggaaagg acccgtaaag tgataatgat tatcatctac atatcacaac 10800 gtgcgtggag gccatcaaac cacgtcaaat aatcaattat gacgcaggta tcgtattaat 10860 tgatctgcat caacttaacg taaaaacaac ttcagacaat acaaatcagc gacactgaat 10920 acggggcaac ctcatgtccc cccccccccc ccccctgcag gcatcgtggt gtcacgctcg 10980 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 11040 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 11100 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 11160 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 11220 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaacac gggataatac cgcgccacat 11280 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 11340 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 11400 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 11460 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 11520 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 11580 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa 11640 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt 11700 51g cttcaagaat tggtcgacga tcttgctgcg ttcggatatt ttcgtggagt tcccgccaca 11760 gacccggatt gaaggcgaga tccagcaact cgcgccagat catcctgtga cggaactttg 11820 gcgcgtgatg actggccagg acgtcggccg aaagagcgac aagcagatca cgcttttcga 11880 cagcgtcgga tttgcgatcg aggatttttc ggcgctgcgc tacgtccgcg accgcgttga 11940 gggatcaagc cacagcagcc cactcgacct tctagccgac ccagacgagc caagggatct 12000 ttttggaatg ctgctccgtc gtcaggcttt ccgacgtttg ggtggttgaa cagaagtcat 12060 tatcgcacgg aatgccaagc actcccgagg ggaaccctgt ggttggcatg cacatacaaa 12120 tggacgaacg gataaacctt ttcacgccct tttaaatatc cgattattct aataaacgct 12180 cttttctctt aggtttaccc gccaatatat cctgtcaaac actgatagtt taaactgaag 12240 gcgggaaacg acaatctgat catgagcgga gaattaaggg agtcacgtta tgacccccgc 12300 cgatgacgcg ggacaagccg ttttacgttt ggaactgaca gaaccgcaac gttgaaggag 12360 ccactcagca agctggtaca agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc 12420 ctctctagag ataatgagca ttgcatgtct aagttataaa aaattaccac atattttttt 12480 tgtcacactt gtttgaagtg cagtttatct atctttatac atatatttaa actttactct 12540 acgaataata taatctatag tactacaata atatcagtgt tttagagaat catataaatg 12600 aacagttaga catggtctaa aggacaattg agtattttga caacaggact ctacagtttt 12660 atctttttag tgtgcatgtg ttctcctttt tttttgcaaa tagcttcacc tatataatac 12720 ttcatccatt ttattagtac atccatttag ggtttagggt taatggtttt tatagactaa 12780 tttttttagt acatctattt tattctattt tagcctctaa attaagaaaa ctaaaactct 12840 attttagttt ttttatttaa taatttagat ataaaataga ataaaataaa gtgactaaaa 12900 attaaacaaa taccctttaa gaaattaaaa aaactaagga aacatttttc ttgtttcgag 12960 tagataatgc cagcctgtta aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc 13020 agcagcgtcg cgtcgggcca agcgaagcag acggcacggc atctctgtcg ctgcctctgg 13080 acccctctcg agagttccgc tccaccgttg gacttgctcc gctgtcggca tccagaaatt 13140 gcgtggcgga gcggcagacg tgagccggca cggcaggcgg cctcctcctc ctctcacggc 13200 accggcagct acgggggatt cctttcccac cgctccttcg ctttcccttc ctcgcccgcc 13260 gtaataaata gacaccccct ccacaccctc tttccccaac ctcgtgttgt tcggagcgca 13320 cacacacaca accagatctc ccccaaatcc acccgtcggc acctccgctt caaggtacgc 13380 cgctcgtcct cccccccccc ccctctctac cttctctaga tcggcgttcc ggtccatggt 13440 tagggcccgg tagttctact tctgttcatg tttgtgttag atccgtgttt gtgttagatc 13500 cgtgctgcta gcgttcgtac acggatgcga cctgtacgtc agacacgttc tgattgctaa 13560 cttgccagtg tttctctttg gggaatcctg ggatggctct agccgttccg cagacgggat 13620 cgatttcatg attttttttg tttcgttgca tagggtttgg tttgcccttt tcctttattt 13680 caatatatgc cgtgcacttg tttgtcgggt catcttttca tgcttttttt tgtcttggtt 13740 gtgatgatgt ggtctggttg ggcggtcgtt ctagatcgga gtagaattct gtttcaaact 13800 acctggtgga tttattaatt ttggatctgt atgtgtgtgc catacatatt catagttacg 13860 aattgaagat gatggatgga aatatcgatc taggataggt atacatgttg atgcgggttt 13920 tactgatgca tatacagaga tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg 13980 ggcggtcgtt cattcgttct agatcggagt agaatactgt ttcaaactac ctggtgtatt 14040 tattaatttt ggaactgtat gtgtgtgtca tacatcttca tagttacgag tttaagatgg 14100 atggaaatat cgatctagga taggtataca tgttgatgtg ggttttactg atgcatatac 14160 atgatggcat atgcagcatc tattcatatg ctctaacctt gagtacctat ctattataat 14220 aaacaagtat gttttataat tattttgatc ttgatatact tggatgatgg catatgcagc 14280 agctatatgt ggattttttt agccctgcct tcatacgcta tttatttgct tggtactgtt 14340 tcttttgtcg atgctcaccc tgttgtttgg tgttacttct gcaggtcgac tctagaggat 14400 ccacc 14405 <210> 4 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - vip3Aa TAQMAN primer <400> 4 caccttcagc aacccgaact a 21 51h <210> 5 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Chemmicall synthesized - vip3Aa TAQMAN primer rev <400> 5 gcttagcctc cacgatcatc tt 22 <210> 6 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - vip3Aa TAQMAN probe <400> 6 gtcctcgtcg ctgcccttca cct 23 <210> 7 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - pmi TAQMAN primer for <400> 7 ccgggtgaat cagcgttt 18 <210> 8 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - pmi TAQMAN primer rev <400> 8 gccgtggcct ttgacagt 18 <210> 9 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - pmi TAQMAN probe <400> 9 tgccgccaac gaatcaccgg 20 51i <210> 10 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - ZmADH-267 TAQMAN primer for <400> 10 gaacgtgtgt tgggtttgca t 21 <210> 11 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - ZmADH-267 TAQMAN primer rev <400> 11 tccagcaatc cttgcacctt 20 <210> 12 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized -ZmADH-267 TAQMAN probe <400> 12 tgcagcctaa ccatgcgcag ggta 24 <210> 13 <211> 2370 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - vip3Aa20 probe <400> 13 atgaacaaga acaacaccaa gctgagcacc cgcgccctgc cgagcttcat cgactacttc 60 aacggcatct acggcttcgc caccggcatc aaggacatca tgaacatgat cttcaagacc 120 gacaccggcg gcgacctgac cctggacgag atcctgaaga accagcagct gctgaacgac 180 atcagcggca agctggacgg cgtgaacggc agcctgaacg acctgatcgc ccagggcaac 240 ctgaacaccg agctgagcaa ggagatcctt aagatcgcca acgagcagaa ccaggtgctg 300.
aacgacgtga acaacaagct ggacgccatc aacaccatgc tgcgcgtgta cctgccgaag 360 atcaccagca tgctgagcga cgtgatgaag cagaactacg ccctgagcct gcagatcgag 420 tacctgagca agcagctgca ggagatcagc gacaagctgg acatcatcaa cgtgaacgtc 480 ctgatcaaca gcaccctgac cgagatcacc ccggcctacc agcgcatcaa gtacgtgaac 540 gagaagttcg aagagctgac cttcgccacc gagaccagca gcaaggtgaa gaaggacggc 600 agcccggccg acatcctgga cgagctgacc gagctgaccg agctggcgaa gagcgtgacc 660 aagaacgacg tggacggctt cgagttctac ctgaacacct tccacgacgt gatggtgggc 720 aacaacctgt tcggccgcag cgccctgaag accgccagcg agctgatcac caaggagaac 780 51j 3fTS
9L1T pe4543 bepoppopqq qbgbobobeq 4gboobboup (÷,TT
05.5ppeo454 op6q.5.6opeo 4ee6peepo.6 op.54;eqq-4.6 obeomee.546 5.5posepqqo beopqq5pob 2p4o445bee ?b5;54q5oe Pob4Pbobbe Etoqb36404 qb444Tepo5 OZOT
oob45ebeob epob244eo3 eop2e2.5uee gpbgbegqoo pbTeDb4obo qp44pobqq4 qeb4ubb4.6P 3314vb3334 43e6bgaeeb u36.45bepop pebqbboobp 33oRfq.4544 5poopeqobb opePeoafree bo4Teee54.5 qeeopbqq.bb qoupb60044 eqs.b4quoeq.
OD'8 p224poboPb qoq..5b5o54.5 ob4obqboPE qeboo4oe2E, obb4e5-45Pe 5.54o5obb45 obbpeobqoo uq4oboeobo oPouPub4ob 31.454o3q4E, Tebobepbob Eqop3ePb4q.
OZL
pep5qbEq14 ppbqp.644Pq 36003oqp4q fiqoqbbabpo ebeebboope 4444Pab4o4 qq.epqq4534 gebosepobb 46opee6q56 6eobeoa6pq ebo4opo550 Tepspqqq.qp boBbi_obobo boop4eepep bepbqbbbpo bqeqpebqqb goobpooboq qbqopubobp eq.44bopubo 3bqeb4op6e oppoPqq4q4 oupgobqqeb obboogeoso 64bEcep6oqb 08t' boobpooqop qopogo45qi s5stopq444 up.54.6oqqqb oboppb4ybo bqqooqq.qop On.
5opfq.obob4 4444.65405e .65pabePopo oPeq.00qP8p psgeqopeqb Dbebooboo5 Tebbi.pb000 qpqbbyoboo .64spepbees 3p54.44q6b3 qeppbqoq.qe POPOPPPOere ep34e3.4465 -2344voogo4 os3o6ppeo6 ro53b4pqqp 46e-evoq.q.bq po4q4pobqo psbobbqq4o boeepoob44 boo56abp.5.6 oqo54o4opb oq.eepTe.64.6 pEce.6.4.4p6.45 qebgb64DE 344q5ogege 6-2.6.6poBoo.6 qepSeobqbe hopoqq5po.6 epsebooqo OZT
eobobbbqeb bqbqobetoo bb4e5oobeo obeop4.6334 eeeebbqe4.6 bge444oe2b q3p64q63bb ouuPpobeob Elthq3pb4P4 3uuse36q.bp oq.peugq.poq opepupobqe t7T <00D'>
agoad Tmd - pazTs3144uics ATTpoTmatm <EZZ>
<OZZ>
Gouanbes TPT0TJT4-rd <ETZ>
VW! <ZTZ>
9LTT <FEZ>
f7T <OTZ>
OLEZ
bpqbeuogeo 52b3.5op5oP qp4gouo6q5 op,Ez oquboop5bo bbou4bqoou poPpobbbeo ofrebqobe5o 4ppeqpq4ae eoeb&epbe5 oq.q.bpuposo oupgqb4e5u bobPbgE3e6 bPuo050663 bebqeop4b6 Pfieebebogq OZZZ
.643b46bebo boo343epo6 op4eobob4f) oPPoobaebo bba6e6q5ob epq.q.opqb45 09TZ boo-2430pp beo44obpou .5.64obPob4o opubeobppb 4poq.pobbo.5 pobbabbbpo OOTZ
pe4bqopopb qooppoppob bobuogPoep opeobeobbo peob?Debb goeepeepo.e 3ee3qpb-405 pbb000bubq a643bPPEmb obeb000bPo qPflebbqopq eogsoqqp2u 0861 op5o65.5b4o o56 5b boee5poo5e .5up54poqpb qooeq.5q5o5 .65PeElqoos5 oopobboopo opoq4obo5e epePoTepoP bepoeqoebb ebbqopsPop up-esopeoeb beboegpeop gepegobboo eoeebeboeb bep54op2oq 4.6oqu.005.2 up.5.6be2.6qb 0081 3p-epeq.6p3o qp6-4bo-e45P booebeuboo 6Pefq.obeeo e63bbo4eog qbepobeoTe 0i7LT
obbobboubb epoup.645ou 4b4opobbee ooPobboepb qlobbobboo epopeb5q.5 ppobopeb epoeepueop Hpubbqloo bubbqoo2eo p.662.6.5pboi. PobuobboPp 0Z91 bEbbqb34po epobpoqeog 4obbobe6oo upp645Dgeb 4a6peposbe bbeeoupobe bp.o3p6o3eo obuq354a6-4. 35Pb353643 oP4obebeeq 54opubqoop up4e6goobo 00s1 obeopubP5o 2boo55e354 po5.6444obb oeupTeb000 oebqopqqop p6ebobuoge 0fq7T
b4bobbbqoe pobgeoegoq bobbouboef) puebobo526 4opopp5oae 45pboobbe5 obeo5ebeb6 gbbpebppbe eoppbqoop6 oqubs.bobbo oeobpobeop boe4o4qaeR
OZET
Do600eb;65 p5oeqobobq oppebeEt4e beebeepopo 44pu534sEce uoopoqp5q6 09z1 peq.bpboeeb pooqqbqboq POEPOPPOOP Deq0e404PP pobebobubp popbbooqbq b4obqobeso eb.64eoPbo.6 boP434-2.64.6 bPbobebqqo buopbbpuo2 bbgbbpoppg 01711 ops6pobee6 qp6Pepobbe boeqbqbbee bqa64.6opuo 4eobeopEop upbsoTebeb 0801 ogq35boqeb qq.boboepob bboobP24ob bPbb45ogpb 4Pbuupobop bEcebop6oe, onT
pabbeebgb.6 uepobosqoe sboop-e-eofm oqq.opeopeo bsbqopopbo obqooTepe-e, fq.b3n3pq.4.6 ubbpbbeebe fibpuoppo44 peobeboput qeo4pobeop epe4o2534e oeb3o.66q33 6664o64o6u soboqbgoos, poe6qopoPb 40344DoElYe 33056pobqo poboopbqob ;6o boo qoPeouqb4b peeobbb455 PbobPobbob PooebePb45 <210> 15 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - MOV3Aa-01-5 primer <400> 15 atgaacaaga acaacaccaa 20 <210> 16 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - MOV3Aa-01-3' primer <400> 16 ctacttgatg ctcacgtcgt ag 22 <210> 17 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 17 acgagcagaa ccaggtgc 18 <210> 18 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 18 ggtgaagaag gacggcag 18 <210> 19 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 19 acctgtcgca agctgctggg 20 <210> 20 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 20 tggacaagct gctgtgtc 18 <210> 21 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 21 tgcaggccga cgagaacag 19 <210> 22 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 22 tgatccagta caccgtgaa 19 <210> 23 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 23 accctgaccc tgtaccag 18 <210> 24 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 24 gtgttgccgc tgatgttg 18 51m <210> 25 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 25 cgtactcggt cttcggct 18 <210> 26 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 26 ctgcaggcca aagccgtt 18 <210> 27 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 27 tcgccgtaga tcacctcg 18 <210> 28 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemcially synthesized <400> 28 gcttgcgaca ggtggtca 18 <210> 29 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 29 ttgctgctgg tctcggtgg 19 in <210> 30 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 30 cgttggcgat cttaaggat 19 <210> 31 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 31 gcaagccatc gattcac 17 <210> 32 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 32 gcaacaccct gaccctg 17 <210> 33 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 33 tctacgacgt gagcatcaag 20 <210> 34 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 34 gtagaagtgc acgatcggg 19 =
<210> 35 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemcially synthesized <400> 35 cggtgctggt ccagttg 17 <210> 36 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Chemcially synthesized - 162INSERT-F2 primer <400> 36 acaccaatga tgcaaatagg c 21 <210> 37 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - VIP_R4 primer <400> 37 gaaggtgttc aggtagaact cgaag 25 <210> 38 <211> 2946 <212> DNA
<213> Artificial Sequence <220>
<223> Chemcially synthesized - vip3Aa20 5' amplicon <400> 38 acaccaatga tgcaaatagg ctgggaatag tctgtctaat agtttgagtg aatcatgtca 60 ctgatagttt aaactgaagg cgggaaacga caatctgatc atgagcggag aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt tttacgtttg gaactgacag aaccgcaacg ttgaaggagc cactcagcaa gctggtacaa gcttgcatgc ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa aactaaggaa 51p acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga cgagtctaac 840 ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga cggcacggca 900 tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg acttgctccg 960 ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac ggcaggcggc 1020 ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc gctccttcgc 1080 tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct ttccccaacc 1140 tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca cccgtcggca 1200 cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc ttctctagat 1260 cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt ttgtgttaga 1320 tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac ctgtacgtca 1380 gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg gatggctcta 1440 gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat agggtttggt 1500 ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc atcttttcat 1560 gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc tagatcggag 1620 tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta tgtgtgtgcc 1680 atacatattc atagttacga attgaagatg atggatggaa atatcgatct aggataggta 1740 tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt cgcttggttg 1800 tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta gaatactgtt 1860 tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat acatcttcat 1920 agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat gttgatgtgg 1980 gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc tctaaccttg 2040 agtacctatc tattataata aacaagtatg ttttataatt attttgatct tgatatactt 2100 ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt catacgctat 2160 ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt gttacttctg 2220 caggtcgact ctagaggatc caccatgaac aagaacaaca ccaagctgag cacccgcgcc 2280 ctgccgagct tcatcgacta cttcaacggc atctacggct tcgccaccgg catcaaggac 2340 atcatgaaca tgatcttcaa gaccgacacc ggcggcgacc tgaccctgga cgagatcctg 2400 aagaaccagc agctgctgaa cgacatcagc ggcaagctgg acggcgtgaa cggcagcctg 2460 aacgacctga tcgcccaggg caacctgaac accgagctga gcaaggagat ccttaagatc 2520 gccaacgagc agaaccaggt gctgaacgac gtgaacaaca agctggacgc catcaacacc 2580 atgctgcgcg tgtacctgcc gaagatcacc agcatgctga gcgacgtgat taagcagaac 2640 tacgccctga gcctgcagat cgagtacctg agcaagcagc tgcaggagat cagcgacaag 2700 ctggacatca tcaacgtgaa cgtcctgatc aacagcaccc tgaccgagat caccccggcc 2760 taccagcgca tcaagtacgt gaacgagaag ttcgaagagc tgaccttcgc caccgagacc 2820 agcagcaagg tgaagaagga cggcagcccg gccgacatcc tggacgagct gaccgagctg 2880 accgagctgg cgaagagcgt gaccaagaac gacgtggacg gcttcgagtt ctacctgaac 2940 accttc 2946 <210> 39 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - CJB179 Primer <400> 39 atgcaaatag gctgggaata gtc 23 <210> 40 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - CTRB3116 Reverse Primer 51q <400> 40 gtaccagctt gctgagtggc t 21 <210> 41 <211> 209 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - CJB134/179 5' amplicon <400> 41 atgcaaatag gctgggaata gtctgtctaa tagtttgagt gaatcatgtc actgatagtt 60 taaactgaag gcgggaaacg acaatctgat catgagcgga gaattaaggg agtcacgtta 120 tgacccccgc cgatgacgcg ggacaagccg ttttacgttt ggaactgaca gaaccgcaac 180 gttgaaggag ccactcagca agctggtac 209 <210> 42 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemcially synthesized - VIP-F3 primer <400> 42 ggtgctgttc gagaagaggt 20 <210> 43 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - PMI_REV1 primer <400> 43 cgatttatca ctctcaatca cat 23 <210> 44 <211> 2577 <212> DNA
<213> Artificial Sequence <220>
<223> Chemcially synthesized - vip3Aa20 3' amplicon <400> 44 ggtgctgttc gagaagaggt acatgagcgg cgccaaggac gtgagcgaga tgttcaccac 60 caagttcgag aaggacaact tctacatcga gctgagccag ggcaacaacc tgtacggcgg 120 cccgatcgtg cacttctacg acgtgagcat caagtaggag ctctagatct gttctgcaca 180 aagtggagta gtcagtcatc gatcaggaac cagacaccag acttttattc atacagtgaa 240 gtgaagtgaa gtgcagtgca gtgagttgct ggtttttgta caacttagta tgtatttgta 300 tttgtaaaat acttctatca ataaaatttc taattcctaa aaccaaaatc caggggtacc 360 51r agcttgcatg cctgcagtgc agcgtgaccc ggtcgtgccc ctctctagag ataatgagca 420 ttgcatgtct aagttataaa aaattaccac atattttttt tgtcacactt gtttgaagtg 480 cagtttatct atctttatac atatatttaa actttactct acgaataata taatctatag 540 tactacaata atatcagtgt tttagagaat catataaatg aacagttaga catggtctaa 600 aggacaattg agtattttga caacaggact ctacagtttt atctttttag tgtgcatgtg 660 ttctcctttt tt.tttgcaaa tagcttcacc tatataatac ttcatccatt ttattagtac 720 atccatttag ggtttagggt taatggtttt tatagactaa tttttttagt acatctattt 780 tattctattt tagcctctaa attaagaaaa ctaaaactct attttagttt ttttatttaa 840 taatttagat ataaaataga ataaaataaa gtgactaaaa attaaacaaa taccctttaa 900 gaaattaaaa aaactaagga aacatttttc ttgtttcgag tagataatgc cagcctgtta 960 aacgccgtcg acgagtctaa cggacaccaa ccagcgaacc agcagcgtcg cgtcgggcca 1020 agcgaagcag acggcacggc atctctgtcg ctgcctctgg acccctctcg agagttccgc 1080 tccaccgttg gacttgctcc gctgtcggca tccagaaatt gcgtggcgga gcggcagacg 1140 tgagccggca cggcaggcgg cctcctcctc ctctcacggc accggcagct acgggggatt 1200 cctttcccac cgctccttcg ctttcccttc ctcgcccgcc gtaataaata gacaccccct 1260 ccacaccctc tttccccaac ctcgtgttgt tcggagcgca cacacacaca accagatctc 1320 ccccaaatcc acccgtcggc acctccgctt caaggtacgc cgctcgtcct cccccccccc 1380 ccctctctac cttctctaga tcggcgttcc ggtccatggt tagggcccgg tagttctact 1440 tctgttcatg tttgtgttag atccgtgttt gtgttagatc cgtgctgcta gcgttcgtac 1500 acggatgcga cctgtacgtc agacacgttc tgattgctaa cttgccagtg tttctctttg 1560 gggaatcctg ggatggctct agccgttccg cagacgggat cgatttcatg attttttttg 1620 tttcgttgca tagggtttgg tttgcccttt tcctttattt caatatatgc cgtgcacttg 1680 tttgtcgggt catcttttca tgcttttttt tgtcttggtt gtgatgatgt ggtctggttg 1740 ggcggtcgtt ctagatcgga gtagaattct gtttcaaact acctggtgga tttattaatt 1800 ttggatctgt atgtgtgtgc catacatatt catagttacg aattgaagat gatggatgga 1860 aatatcgatc taggataggt atacatgttg atgcgggttt tactgatgca tatacagaga 1920 tgctttttgt tcgcttggtt gtgatgatgt ggtgtggttg ggcggtcgtt cattcgttct 1980 agatcggagt agaatactgt ttcaaactac ctggtgtatt tattaatttt ggaactgtat 2040 gtgtgtgtca tacatcttca tagttacgag tttaagatgg atggaaatat cgatctagga 2100 taggtataca tgttgatgtg ggttttactg atgcatatac atgatggcat atgcagcatc 2160 tattcatatg ctctaacctt gagtacctat ctattataat aaacaagtat gttttataat 2220 tattttgatc ttgatatact tggatgatgg catatgcagc agctatatgt ggattttttt 2280 agccctgcct tcatacgcta tttatttgct tggtactgtt tcttttgtcg atgctcaccc 2340 tgttgtttgg tgttacttct gcagggatcc ccgatcatgc aaaaactcat taactcagtg 2400 caaaactatg cctggggcag caaaacggcg ttgactgaac tttatggtat ggaaaatccg 2460 tccagccagc cgatggccga gctgtggatg ggcgcacatc cgaaaagcag ttcacgagtg 2520 cagaatgccg ccggagatat cgtttcactg cgtgatgtga ttgagagtga taaatcg 2577 <210> 45 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> 5' junction sequence between corn genome and insert DNA
<400> 45 tgaatcatgt cactgatagt 20 <210> 46 =
<211> 1088 <212> DNA
<213> Artificial Sequence <220>
<223> 5' flanking sequence 51s <220>
<221> misc_feature <222> (1)..(236) <223> 5' flanking sequence <400> 46 tcctgtgttg ttggaacaga cttctgtctc ttctggtgat cataaatatt taaatgaacc 60 agttgtgttg gaaaatgttg ttttcttttg tctctagact ggaaagcgga gttctcgtca 120 acacggttct ttcaactagg gatgaaagtg gtaatccgaa ttgttagtac aaatttaata 180 ttttaaaata gatatgtata aaattttatg ttgatctttt ttatgttatc aagcacatta 240 gtataaatta gtataaatat gaataaaata ttacataaaa tgttttatgt attatttggt 300 ccctacaaca taaatagttg aaaaaattac taaatttgtt ttcgaatcta tatcgaagtt 360 tatatctatt atttaagaaa aatataggat gaaaaggttt atcttttatg aatctttaca 420 agctggatct tataaacaag aaaataaatt tatattgtag attttatatc ctatttattc 480 gcaatcaaag aaaagcgact aaaaaactga ttaccgagta aatactgttt ccaaccgttt 540 tcgtccctac tatcaacgcc ttctcccaac cgcagtcgat ctgtccgtct gtatcaggcg 600 cagcggcacc cctgctgttc gactatctag accatagaat attttaggta tacaataatt 660 ttagttccac gctagaacat tttagttaga ataataacaa gatttgctat tgatgtagga 720 ctcgcccgtc actgtctaaa aaagcattct gtcggtctta ttctttaggc atcagcgggt 780 gtactatctc atttttccta tcatattcct cagtactctg ttaagtataa atggtctatt 840 ttacatgatg aactaataaa actaattaag gatcctaact ttttgtgaag gtaatttgga 900 tcattatgca ttaccatcct acgtatacct gctgcagcag catctgcgta agcacagcct 960 agatatatgc ttctgtgtgg actgaaagga gactttgttt atcaattagt atactcccaa 1020 aaaactgatg acaccaatga tgcaaatagg ctgggaatag tctgtctaat agtttgagtg 1080 aatcatgt 1088 <210> 47 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> 3' junction sequence between insert DNA and corn genome <400> 47 aaacgtccgc catggtctga 20 <210> 48 <211> 1189 <212> DNA
<213> Artificial Sequence <220>
<223> 3' flanking sequence <400> 48 catggtctga aggcaacaga taaggcatac tgggccttgt ggtagttgtt ttactgggcc 60 tttttgtatg atctataaaa ttcactggga tcaacccgga gaggaatggc agcagatgca 120 gtccccaggg tcctccgtcg ccgcctgagc acccggcacc cgcgctgaac cggagaggga 180 cgcgcggacg ccgtgcagct ggtgcggagg gggctgtggc agatgaggat gagacgcgta 240 cgtggctggg aaggccagca ggccaccggg tcttcgtcca gcccggcgcg agtggacagg 300 actagagatg gcaacggtta caaacccgct gggttttacc gtcccaaacc cgtacccgtg 360 aaaaatatct atgcccatta aaaaacccgt acccatgacg ggtttgagat tttgcccaaa 420 cccgtaccca tcgggttaac gggtacccat gggttacccg cgggtttcat ctccaatata 480 cctgttcttc tcataatcaa taagtatcgt aatgattaat gatatcatga tccaaaatct 540 atgtaatgaa caacgagttc atgatttggt ataaaaatta ttagtagaga gaatgaaata 600 51t caaataataa gttgtataat taagtgacct tgcactaagt tatccatcca tcacatatat 660 aacgctagta aaaactataa tatcaagcaa gcaacactct caccgactac tgatacattc 720 accaattgat aaaaaatatg aagtaaataa ggaataacaa gtttgttgtt cgtttataaa 780 ataaaatgac aatatgcact aggtttggtc gggtttaaaa aacccacggg ttcacgggtt 840 tgggtactat aggaacaaac ccgtacccat aaacccattg ggtacagatt tatgcccgtt 900 aacaaaccca tgggtatgaa aattgaccca aacctatacc ctaatggggt aaaaacccat 960 cgggtttcgg atttcgggta cccattgcca tctctagaca ggacaacctc ggccggtcct 1020 gtatgtaggc caccagcatc ggccagttgg tacatccagc cggggtcagg tcacttttac 1080 tcgtctcaat cagacaatca ccgtccacca acgaacgcca acgttgtcac ttgtcaggtc 1140 ggttgagact tgtatttttt tttgtcctcc gtaaaaatcg gttcaccag 1189 <210> 49 <211> 10579 <212> DNA
<213> Artificial Sequence <220>
<223> Vip3Aa20 Event MIR162 inert and flanking sequences <220>
<221> misc feature <222> (1)..(1088) <223> 5' flanking sequence of event MIR162 <400> 49 tcctgtgttg ttggaacaga cttctgtctc ttctggtgat cataaatatt taaatgaacc 60 agttgtgttg gaaaatgttg ttttcttttg tctctagact ggaaagcgga gttctcgtca 120 acacggttct ttcaactagg gatgaaagtg gtaatccgaa ttgttagtac aaatttaata 180 ttttaaaata gatatgtata aaattttatg ttgatctttt ttatgttatc aagcacatta 240 gtataaatta gtataaatat gaataaaata ttacataaaa tgttttatgt attatttggt 300 ccctacaaca taaatagttg aaaaaattac taaatttgtt ttcgaatcta tatcgaagtt 360 tatatctatt atttaagaaa aatataggat gaaaaggttt atcttttatg aatctttaca 420 agctggatct tataaacaag aaaataaatt tatattgtag attttatatc ctatttattc 480 gcaatcaaag aaaagcgact aaaaaactga ttaccgagta aatactgttt ccaaccgttt 540 tcgtccctac tatcaacgcc ttctcccaac cgcagtcgat ctgtccgtct gtatcaggcg 600 cagcggcacc cctgctgttc gactatctag accatagaat attttaggta tacaataatt 660 ttagttccac gctagaacat tttagttaga ataataacaa gatttgctat tgatgtagga 720 ctcgcccgtc actgtctaaa aaagcattct gtcggtctta ttctttaggc atcagcgggt 780 gtactatctc atttttccta tcatattcct cagtactctg ttaagtataa atggtctatt 840 ttacatgatg aactaataaa actaattaag gatcctaact ttttgtgaag gtaatttgga 900 tcattatgca ttaccatcct acgtatacct gctgcagcag catctgcgta agcacagcct 960 agatatatgc ttctgtgtgg actgaaagga gactttgttt atcaattagt atactcccaa 1020 aaaactgatg acaccaatga tgcaaatagg ctgggaatag tctgtctaat agtttgagtg 1080 aatcatgtca ctgatagttt aaactgaagg cgggaaacga caatctgatc atgagcggag 1140 aattaaggga gtcacgttat gacccccgcc gatgacgcgg gacaagccgt tttacgtttg 1200 gaactgacag aaccgcaacg ttgaaggagc cactcagcaa gctggtacaa gcttgcatgc 1260 ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 1320 agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 1380 tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 1440 tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 1500 gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 1560 ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 1620 , gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 1680 agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 1740 taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 1800 aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 1860 cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 1920 51u ATS
(pops eobeebqooq pobboboobb obbbpope4b qopoubqopo epeeobbo62 oqppepooep opEg buobboopob pooebbqopp OPPOOPOPPO 4vb436u663 op6254ob4o beebubpbeb ogzg opo5poqpflp bbqopqpoge oqqoposbo 6.655qop6bp bo?bo56pe? fveop6e5e5 OZZS
qop4e6qopp qbqbobEibpp .6400pboopo Mopeopepq 4obobepopp ogPooPbeop ogTg Pqop5.6.265-4 DOPPOPPOeP opeoubbp5o eqoepoqeD2 43.6bopuosre bP5oe6bPP5 OOTS
4DOPOqq.E.60 qupobepobb bPubqbooPo eqbPooTebq bouqbPbooe bPeboobeb opog qobeepe6o5 boTeoqqbuo o5Poqeobbo bbopbbpeop ofq.bopqbqo po5bppop2o 086D' Moss.5qbab bobbooPoeo Deb6q6Dp4o o5oee6ppoe popepobbup bb46po5p.66 oz61 4opePou55p .6.5pbogeob2 obbosebe55 qboTeoPpob uoq2pqqobb ob.e.booPoob O98t.
45oqp5go5E. popebe66ep oppobpbqop pboepobeq 36qa5.4a5p6 obob4popqo bebpeqbqop vb4000po4e bqpoboobpo eebebouboo bbeobqoobb 44go5bo-eso opLp geboopopbq poq400e6pb ofleoqe54bo 6b6qopoo6q posqpqbobb oPboeboPeb obo5pbqopo eoboo2qbe6 cobbebobpo bb2bE,4.6.6p 2EPP.6PPOPP bqoppboqsb p6pabopeo5 eobpoeboug oqqopuoobo op6q56-eboe go6o6qp=e BPP64ebeeb 09;17 eepooqqoe bo4a6ePope oqe.64boe4b uboeuboop4 4b4bo42oe2 oeeoopopqo 00Sf, P4o4eppo6p bobebpopeb booqb4,64ob 4obeeou55q popbo5Elopq o4e6.455e6o OM
beb41.obepe bbueoeba4b bepoe4peeb eabeeb4oL2 epobbpboeq 54.65uubqob =
ogEp 45oopoTeo5 eppboepobe oTe5ebo443 5boTeeq4bo boepobbboo Beeqobb-e56 OZED, 46o4ebqebe eopboebbeb oebobeobbb eebq.662eoD boeqopeboo oe2o5po4.43 ogzp oep?vo6pbq. opoe5oo.540 oqeope645o Bpoqq6p65p 65ree5eb6ee pep.54qopob oozp eboeeb4234 eobeoDeoeq opbo4epeLo obbqoobbbq o64obeeo5o qbgoovoo-eb opTp qopoPbqopq 400bbeoopb Beofy4poo6o op5qab.450.4 eLgooqqoPu os-4545op23 080f7 6664bbebob eobelobeooe beeb4boeeb ebbeepoeoq ebqobebobe poElooebeeb ozop qopobo5po6 po55oqq540 oppopeo6E6 456Te5q5ou boPooqqope pee6qoaeqo 44bv50qq05 boebbqboPb OPEZEPOOPE, 4bobp6Pe5o E6qpbebooe b4obebooeb 4obuloebbq opqeDeboob boopbuo65o e55efres,5.4 6beeobpobp oo2beboopo oboqq.oppbq 35Pbeutoq.4 bpuficeboppb gEoP4.6epog p3b35pooR4 pobbooppeo ogLE
4pbsboopbq 000P0bPOPP oqe54=4.63 pbqbaepoq soqPop554o 6peoe5o5eo onE
4eEre55P3bq. ofipoElppobp bqoppq.5p6o qpbpob433.6 pbqopoboeq. oeabsob-evb qubqbopbob ebqobqeobe opeoqebepb op5qpoeq5q bobobqobge DOPOPPOT20 009E 363 636 epepopPb4b oebopabqob 4b6eoppe5u obebopeoob ogpbeeqqop 0D,sE
4eBe5bepob ebqobebooe oeebqpopo bbbpopobog ebqoopboep 64006po55o ee6gE3El0p bbqobupoff, 3bP34-e0e53 pp6qobqob-2 o5reoppu5ep bqopqpfiebo ozpE
ebbqopoebq opp6o65obb opeoe5oop5 ppoT4o4e.6.4 eoep8qpo4p 3pbbpuo4eo bbooPpobai. 406,53-2q34P ob5oeuo4go eqopElogpoq qo6eboo54o opbaboopeo HEE
5rebqp6usoo Poespepbee opPb4popeo oqpb5p5eqo 4op5pq5Seo 6qpq4o-e-44.5 opzE
46644q64qb 4pooppgabq Pbo45qqq4o 44q640e466 .4435.4qmegq Teqoboe4eo ogTE
.4.4005y4opob eqq-44;q4pb 5q5qe.4.2qa5 eobeobTeqe ob6qe5Te5b .44oeqeqPbq OZTE
40Tebqq4qP 44eeqPq4qq- bqPqbPPoPP PqPP4q4Pq 04eq0DP4be 54400Pu404 ofiqvq.pogge gogeo6ep54 egPobbgebq pougeT2o64 .ebqosqqq..45 6646qpb;45 4eoeqpq.65p qe5.6pq34u6 oqpqpepfilq. pfibTefiepqq 4bubopqqbe 4Poqqoqeop Of763 4P04545454 54P4540?Pb 54444Pe44P 4'44454664 00-240ePPoq 44b40e4P'eb oggz Pqbpbboqpb eqoqq.604qe ogmbogfibob 6b 655 6q6.4e5qebq 54-4654.4.63 44.5444qqob qe6pb2ougP 4po54pb4ou 4.4q4bb6o5q ?Eqqbquopq Eqbbe4u66p ogLz qoqeboTeqp se6642.664P 66eebqqp eboeqqbp4p 34Teqpopqe op6m6gbqbq OOLZ
e4b43qP6bq 44gesqqe4q Tebb4b.6qoo 2qopeuom 64o44,22bug bPbbo4pbuq OD'9Z
0T45046b06 5.54;bbq046 5454s5qP5q. 54465qq046 44T44qqq0.6 Te0q4qqoqe 34Bbb34644 4644peob4b opEquge4ep 04q4e344op 44;qcoo5qq. 45544qb6be ozgz 4eo64q6o4q. .4.6qqqqqq4.4 pf)Teogqq.?f, oqP656oe5e o5oogqboo5 sqoqo.5.5.4p6 86400gee65 EI.Eqqq34044 qb4bepob4q oeeqpb4qeb 434g5p2ou5 e3q5op4.6qo oopz opbo5Te55o popgboqq6o 5eqo5qp545 poqefreqqbq 544q5.4630.4 E5yegq5;644 OPEZ
464R04q540 440P40q46P 4E6000555P 4m6b4e3o46 600qgbo65o 4e6eqp4044 oezz oopqoqogoo 3003033033 oq=4.6ogob pobopq6,6?e oqqa5oo4po ea65o46poo oz epogeupoop op4o4p6poo pppeoppeop oeobobebbo 446.4q6,4534 poee000pqq.
ogTz 4oqopouopo oq000popo2 6.eqe-224e-24 boo5oop6o4 opqqopoqq; oBoqqopqob OOTZ
opuoopqqqo 344-ebel6bbo si.obobboo pon5ouoq.34 ooqoogoogo obbobbeobb ovoz opobboo6p5 4bopbpo55o 6pb5ob54bo 6.4.4ppp5e3o Teobboq54o boogoEqqop 654463ovo3 qp5oogq6pb eboqog000p PE64340064 06345434o4 pobbopobbo gaacctgcag ctggacagct tcagcaccta ccgcgtgtac ttcagcgtga gcggcgacgc 5460 caacgtgcgc atccgcaact cccgcgaggt gctgttcgag aagaggtaca tgagcggcgc 5520 caaggacgtg agcgagatgt tcaccaccaa gttcgagaag gacaacttct acatcgagct 5580 gagccagggc aacaacctgt acggcggccc gatcgtgcac ttctacgacg tgagcatcaa 5640 gtaggagctc tagatctgtt ctgcacaaag tggagtagtc agtcatcgat caggaaccag 5700 acaccagact tttattcata cagtgaagtg aagtgaagtg cagtgcagtg agttgctggt 5760 ttttgtacaa cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa 5820 ttcctaaaac caaaatccag gggtaccagc ttgcatgcct gcagtgcagc gtgacccggt 5880 cgtgcccctc tctagagata atgagcattg catgtctaag ttataaaaaa ttaccacata 5940 ttttttttgt cacacttgtt tgaagtgcag tttatctatc tttatacata tatttaaact 6000 ttactctacg aataatataa tctatagtac tacaataata tcagtgtttt agagaatcat 6060 ataaatgaac agttagacat ggtctaaagg acaattgagt attttgacaa caggactcta 6120 cagttttatc tttttagtgt gcatgtgttc tccttttttt ttgcaaatag cttcacctat 6180 ataatacttc atccatttta ttagtacatc catttagggt ttagggttaa tggtttttat 6240 agactaattt ttttagtaca tctattttat tctattttag cctctaaatt aagaaaacta 6300 aaactctatt ttagtttttt tatttaataa tttagatata aaatagaata aaataaagtg 6360 actaaaaatt aaacaaatac cctttaagaa attaaaaaaa ctaaggaaac atttttcttg 6420 tttcgagtag ataatgccag cctgttaaac gccgtcgacg agtctaacgg acaccaacca 6480 gcgaaccagc agcgtcgcgt cgggccaagc gaagcagacg gcacggcatc tctgtcgctg 6540 cctctggacc cctctcgaga gttccgctcc accgttggac ttgctccgct gtcggcatcc 6600 agaaattgcg tggcggagcg gcagacgtga gccggcacgg caggcggcct cctcctcctc 6660 tcacggcacc ggcagctacg ggggattcct ttcccaccgc tccttcgctt tcccttcctc 6720 gcccgccgta ataaatagac accccctcca caccctcttt ccccaacctc gtgttgttcg 6780 gagcgcacac acacacaacc agatctcccc caaatccacc cgtcggcacc tccgcttcaa 6840 ggtacgccgc tcgtcctccc cccccccccc tctctacctt ctctagatcg gcgttccggt 6900 ccatggttag ggcccggtag ttctacttct gttcatgttt gtgttagatc cgtgtttgtg 6960 ttagatccgt gctgctagcg ttcgtacacg gatgcgacct gtacgtcaga cacgttctga 7020 ttgctaactt gccagtgttt ctctttgggg aatcctggga tggctctagc cgttccgcag 7080 acgggatcga tttcatgatt ttttttgttt cgttgcatag ggtttggttt gcccttttcc 7140 tttatttcaa tatatgccgt gcacttgttt gtcgggtcat cttttcatgc ttttttttgt 7200 cttggttgtg atgatgtggt ctggttgggc ggtcgttcta gatcggagta gaattctgtt 7260 tcaaactacc tggtggattt attaattttg gatctgtatg tgtgtgccat acatattcat 7320 agttacgaat tgaagatgat ggatggaaat atcgatctag gataggtata catgttgatg 7380 cgggttttac tgatgcatat acagagatgc tttttgttcg cttggttgtg atgatgtggt 7440 gtggttgggc ggtcgttcat tcgttctaga tcggagtaga atactgtttc aaactacctg 7500 gtgtatttat taattttgga actgtatgtg tgtgtcatac atcttcatag ttacgagttt 7560 aagatggatg gaaatatcga tctaggatag gtatacatgt tgatgtgggt tttactgatg 7620 ' catatacatg atggcatatg cagcatctat tcatatgctc taaccttgag tacctatcta 7680 ttataataaa caagtatgtt ttataattat tttgatcttg atatacttgg atgatggcat 7740 atgcagcagc tatatgtgga tttttttagc cctgccttca tacgctattt atttgcttgg 7800 tactgtttct tttgtcgatg ctcaccctgt tgtttggtgt tacttctgca gggatccccg 7860 atcatgcaaa aactcattaa ctcagtgcaa aactatgcct ggggcagcaa aacggcgttg 7920 actgaacttt atggtatgga aaatccgtcc agccagccga tggccgagct gtggatgggc 7980 gcacatccga aaagcagttc acgagtgcag aatgccgccg gagatatcgt ttcactgcgt 8040 gatgtgattg agagtgataa atcgactctg ctcggagagg ccgttgccaa acgctttggc 8100 gaactgcctt tcctgttcaa agtattatgc gcagcacagc cactctccat tcaggttcat 8160 ccaaacaaac acaattctga aatcggtttt gccaaagaaa atgccgcagg tatcccgatg 8220 gatgccgccg agcgtaacta taaagatcct aaccacaagc cggagctggt ttttgcgctg 8280 acgcctttcc ttgcgatgaa cgcgtttcgt gaattttccg agattgtctc cctactccag 8340 ccggtcgcag gtgcacatcc ggcgattgct cactttttac aacagcctga tgccgaacgt 8400 ttaagcgaac tgttcgccag cctgttgaat atgcagggtg aagaaaaatc ccgcgcgctg 8460 gcgattttaa aatcggccct cgatagccag cagggtgaac cgtggcaaac gattcgttta 8520 atttctgaat tttacccgga agacagcggt ctgttctccc cgctattgct gaatgtggtg 8580 aaattgaacc ctggcgaagc gatgttcctg ttcgctgaaa caccgcacgc ttacctgcaa 8640 ggcgtggcgc tggaagtgat ggcaaactcc gataacgtgc tgcgtgcggg tctgacgcct 8700 aaatacattg atattccgga actggttgcc aatgtgaaat tcgaagccaa accggctaac 8760 cagttgttga cccagccggt gaaacaaggt gcagaactgg acttcccgat tccagtggat 8820 gattttgcct tctcgctgca tgaccttagt gataaagaaa ccaccattag ccagcagagt 8880 51w gccgccattt tgttctgcgt cgaaggcgat gcaacgttgt ggaaaggttc tcagcagtta cagcttaaac cgggtgaatc agcgtttatt gccgccaacg aatcaccggt gactgtcaaa ggccacggcc gtttagcgcg tgtttacaac aagctgtaag agcttactga aaaaattaac atctcttgct aagctgggag ctcgatccgt cgacctgcag atcgttcaaa catttggcaa taaagtttct taagattgaa tcctgttgcc ggtcttgcga tgattatcat ataatttctg ttgaattacg ttaagcatgt aataattaac atgtaatgca tgacgttatt tatgagatgg gtttttatga ttagagtccc gcaattatac atttaatacg cgatagaaaa caaaatatag cgcgcaaact aggataaatt atcgcgcgcg gtgtcatcta tgttactaga tccccgggtc 9360 tagacaattc agtacattaa aaacgtccgc catggtctga aggcaacaga taaggcatac tgggccttgt ggtagttgtt ttactgggcc tttttgtatg atctataaaa ttcactggga tcaacccgga gaggaatggc agcagatgca gtccccaggg tcctccgtcg ccgcctgagc acccggcacc cgcgctgaac cggagaggga cgcgcggacg ccgtgcagct ggtgcggagg gggctgtggc agatgaggat gagacgcgta cgtggctggg aaggccagca ggccaccggg 9660 tcttcgtcca gcccggcgcg agtggacagg actagagatg gcaacggtta caaacccgct gggttttacc gtcccaaacc cgtacccgtg aaaaatatct atgcccatta aaaaacccgt acccatgacg ggtttgagat tttgcccaaa cccgtaccca tcgggttaac gggtacccat gggttacccg cgggtttcat ctccaatata cctgttcttc tcataatcaa taagtatcgt aatgattaat gatatcatga tccaaaatct atgtaatgaa caacgagttc atgatttggt ataaaaatta ttagtagaga gaatgaaata caaataataa gttgtataat taagtgacct 10020 tgcactaagt tatccatcca tcacatatat aacgctagta aaaactataa tatcaagcaa 10080 gcaacactct caccgactac tgatacattc accaattgat aaaaaatatg aagtaaataa 10140 ggaataacaa gtttgttgtt cgtttataaa ataaaatgac aatatgcact aggtttggtc 10200 gggtttaaaa aacccacggg ttcacgggtt tgggtactat aggaacaaac ccgtacccat 10260 aaacccattg ggtacagatt tatgcccgtt aacaaaccca tgggtatgaa aattgaccca 10320 aacctatacc ctaatggggt aaaaacccat cgggtttcgg atttcgggta cccattgcca 10380 tctctagaca ggacaacctc ggccggtcct gtatgtaggc caccagcatc ggccagttgg 10440 tacatccagc cggggtcagg tcacttttac tcgtctcaat cagacaatca ccgtccacca 10500 acgaacgcca acgttgtcac ttgtcaggtc ggttgagact tgtatttttt tttgtcctcc 10560 gtaaaaatcg gttcaccag <210> 50 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - FE1002 primer <400> 50 cgtgactccc ttaattctcc gct 23 <210> 51 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - FE1003 primer <400> 51 gatcagattg tcgtttcccg cctt 24 <210> 52 <211> 24 51x <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - 51004 primer <400> 52 gattgtcgtt tcccgccttc agtt 24 <210> 53 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - 162_DW_Conf3 primer <400> 53 cctgtgttgt tggaacagac ttctgtc 27 <210> 54 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - FE0900 primer <400> 54 ggctccttca acgttgcggt tctgtc 26 <210> 55 <211> 1230 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - 5' PCR amplicon <400> 55 cctgtgttgt tggaacagac ttctgtctct tctggtgatc ataaatattt aaatgaacca 60 gttgtgttgg aaaatgttgt tttcttttgt ctctagactg gaaagcggag ttctcgtcaa 120 cacggttctt tcaactaggg atgaaagtgg taatccgaat tgttagtaca aatttaatat 180 tttaaaatag atatgtataa aattttatgt tgatcttttt tatgttatca agcacattag 240 tataaattag tataaatatg aataaaatat tacataaaat gttttatgta ttatttggtc 300 cctacaacat aaatagttga aaaaattact aaatttgttt tcgaatctat atcgaagttt 360 atatctatta tttaagaaaa atataggatg aaaaggttta tcttttatga atctttacaa 420 gctggatctt ataaacaaga aaataaattt atattgtaga ttttatatcc tatttattcg 480 caatcaaaga aaagcgacta aaaaactgat taccgagtaa atactgtttc caaccgtttt 540 cgtccctact atcaacgcct tctcccaacc gcagtcgatc tgtccgtctg tatcaggcgc 600 agcggcaccc ctgctgttcg actatctaga ccatagaata ttttaggtat acaataattt 660 tagttccacg ctagaacatt ttagttagaa taataacaag atttgctatt gatgtaggac 720 tcgcccgtca ctgtctaaaa aagcattctg tcggtcttat tctttaggca tcagcgggtg 780 tactatctca tttttcctat catattcctc agtactctgt taagtataaa tggtctattt 840 tacatgatga actaataaaa ctaattaagg atcctaactt tttgtgaagg taatttggat 900 51y cattatgcat taccatccta cgtatacctg ctgcagcagc atctgcgtaa gcacagccta 960 gatatatgct tctgtgtgga ctgaaaggag actttgttta tcaattagta tactcccaaa 1020 aaactgatga caccaatgat gcaaataggc tgggaatagt ctgtctaata gtttgagtga 1080 atcatgtcac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 1140 attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 1200 aactgacaga accgcaacgt tgaaggagcc 1230 <210> 56 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - 162_GW 3_F1 primer <400> 56 tctcttgcta agctgggagc tcgatccg 28 <210> 57 <211> 28 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - 162_GW 3_F2 primer <400> 57 aagattgaat cctgttgccg gtcttgcg 28 <210> 58 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized162_3'GW R1 primer <400> 58 ctggtgaacc gatttttacg gagg 24 <210> 59 <211> 1518 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - 3' PCR amplicon <400> 59 tctcttgcta agctgggagc tcgatccgtc gacctgcaga tcgttcaaac atttggcaat 60 aaagtttctt aagattgaat cctgttgccg gtcttgcgat gattatcata taatttctgt 120 tgaattacgt taagcatgta ataattaaca tgtaatgcat gacgttattt atgagatggg 180 tttttatgat tagagtcccg caattataca tttaatacgc gatagaaaac aaaatatagc 240 gcgcaaacta ggataaatta tcgcgcgcgg tgtcatctat gttactagat ccccgggtct 300 agacaattca gtacattaaa aacgtccgcc atggtctgaa ggcaacagat aaggcatact 360 51z gggccttgtg gtagttgttt tactgggcct ttttgtatga tctataaaat tcactgggat 420 caacccggag aggaatggca gcagatgcag tccccagggt cctccgtcgc cgcctgagca 480 cccggcaccc gcgctgaacc ggagagggac gcgcggacgc cgtgcagctg gtgcggaggg 540 ggctgtggca gatgaggatg agacgcgtac gtggctggga aggccagcag gccaccgggt 600 cttcgtccag cccggcgcga gtggacagga ctagagatgg caacggttac aaacccgctg 660 ggttttaccg tcccaaaccc gtacccgtga aaaatatcta tgcccattaa aaaacccgta 720 cccatgacgg gtttgagatt ttgcccaaac ccgtacccat cgggttaacg ggtacccatg 780 ggttacccgc gggtttcatc tccaatatac ctgttcttct cataatcaat aagtatcgta 840 atgattaatg atatcatgat ccaaaatcta tgtaatgaac aacgagttca tgatttggta 900 taaaaattat tagtagagag aatgaaatac aaataataag ttgtataatt aagtgacctt 960 gcactaagtt atccatccat cacatatata acgctagtaa aaactataat atcaagcaag 1020 caacactctc accgactact gatacattca ccaattgata aaaaatatga agtaaataag 1080 gaataacaag tttgttgttc gtttataaaa taaaatgaca atatgcacta ggtttggtcg 1140 ggtttaaaaa acccacgggt tcacgggttt gggtactata ggaacaaacc cgtacccata 1200 aacccattgg gtacagattt atgcccgtta acaaacccat gggtatgaaa attgacccaa 1260 acctataccc taatggggta aaaacccatc gggtttcgga tttcgggtac ccattgccat 1320 ctctagacag gacaacctcg gccggtcctg tatgtaggcc accagcatcg gccagttggt 1380 acatccagcc ggggtcaggt cacttttact cgtctcaatc agacaatcac cgtccaccaa 1440 cgaacgccaa cgttgtcact tgtcaggtcg gttgagactt gtattttttt ttgtcctccg 1500 taaaaatcgg ttcaccag 1518 <210> 60 <211> 20 <212> DNA
,<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 60 ttcacgggag actttatctg 20 <210> 61 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 61 ccgattcatt aatgcag 17 <210> 62 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 62 acgtaaaacg gcttgtc 17 51aa <210> 63 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 63 gtttaaactg aaggcgg 17 <210> 64 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 64 aataatatca ctctgtacat cc 22 <210> 65 <211> 15 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 65 gttgtaaaac gacgg 15 <210> 66 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 66 taggcacccc aggcttta 18 <210> 67 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 67 aattgaattt agcggccg 18 lbb <210> 68 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 68 ggtccctaca acataaatag 20 <210> 69 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 69 ttcgtcccta ctatcaacgc 20 <210> 70 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 70 ctttaggcat cagcgggt 18 <210> 71 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 71 agcatctgcg taagcaca 18 <210> 72 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 72 ctgatgacac caatgatgc 19 51cc <210> 73 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 73 gatcagattg tcgtttccc 19 <210> 74 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 74 gcatcattgg tgtcatcag 19 <210> 75 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 75 tgtgcttacg cagatgct 18 <210> 76 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 76 acccgctgat gcctaaag 18 <210> 77 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 77 gcgttgatag tagggacgaa 20 51dd <210> 78 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 78 ctatttatgt tgtagggacc 20 <210> 79 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 79 ctagactgga aagcggag 18 <210> 80 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 80 ccactttcat ccctagttg 19 <210> 81 <211> 17 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 81 gattgaatcc tgttgcc 17 <210> 82 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 82 tctcataaat aacgtcatgc 20 5lee <210> 83 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 83 tctgtggata accgtattac 20 <210> 84 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 84 agtaacatag atgacaccgc 20 <210> 85 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 85 ccagtgtgct ggaattcg 18 <210> 86 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 86 ccagtgtgat ggatatctgc 20 <210> 87 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 87 ccagtgtgct ggaattcg 18 51ff <210> 88 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 88 ccagtgtgat ggatatctgc 20 <210> 89 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 89 gtgtgctgga attcgccctt 20 <210> 90 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 90 tatctgcaga attcgccctt 20 <210> 91 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 91 gtgtgctgga attcgccctt 20 <210> 92 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 92 tatctgcaga attcgccctt 20 51gg <210> 93 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 93 gtgtgctgga attcgccctt 20 <210> 94 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 94 tatctgcaga attcgccctt 20 <210> 95 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 95 gtgtgctgga attcgccctt 20 <210> 96 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 96 ggtcttgcga tgattatc 18 <210> 97 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 97 gagaggaatg gcagcaga 18 51hh <210> 98 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 98 catgacgggt ttgagatt 18 <210> 99 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 99 aatctcaaac ccgtcatg 18 <210> 100 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 100 tctgctgcca ttcctctc 18 <210> 101 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized <400> 101 gatcaacccg gagaggaat 19 <210> 102 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - b05104h sequenceing primer <400> 102 ccatgacggg tttgagat 18 51ii <210> 103 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - b05105c sequenceing primer <400> 103 caaccgacct gacaagtgac 20 <210> 104 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - b05105e sequenceing primer <400> 104 atctcaaacc cgtcatgg 18 <210> 105 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Chemically synthesized - b05105f sequencing primer <400> 105 attcctctcc gggttgatc 19 <210> 106 <211> 51328 <212> DNA
<213> Zea mays <220>
<221> misc feature <222> (168-6)..(3338) <223> opie2 marker <220>
<221> misc feature <222> (254-5-5)..(25512) <223> Maize genomic sequence displaced by MIR162 heterologous DNA.
<220>
<221> misc feature <222> (43275)..(45086) <223> gag marker <400> 106 agatctagag attcgtcatt tgtagaaagg atagaaaggt gattgctagg aagttagggg 60 51jj caaaatgcaa gggtgataaa acttgcattt ggatccctaa ggaaattgtg actaaccttg 120 taggacccaa caagagttgg gtacctaagt cccaagccta aatttgcctt gcaggtttat 180 gcatccgggg gttcaagctg gattattgat agcggatgca caaaccatat gacgggggag 240 aagaagatgt tcacctccta tgtcaagaat aaggattccc aagattcaat tatattcggt 300 gatgggaatc aaggcaaggt aaaagggtta ggtaaaattg caatttctaa tgagcactcc 360 atatctaatg tgtttttagt agagtctctc agatataatt tgctatctgt tagtcaatta 420 tgcaacatgg ggtataactg tctatttacc aatgtagatg tgtctgtctt tagaagaagt 480 gatggttcac tagcttttaa gggtgtatta gacggcaaac tttatttagt tgattttgca 540 aaagaagagg ccggtctaga tgcatgctta atagctaaga ctagcatggg ctggctgtgg 600 catcgccgct tagcacatgt ggggatgaag aaccttcaca agcttctaaa gggagaacac 660 gtgataggtt tgactaatgt gcaattcgaa aaagatagac cttgtgcagc ttgtcaagca 720 gggaaacagg tgggaagcgc tcatcacacc aaaaatgtga tgacaacatc aagacccctg 780 gagctgctac atatggacct cttcggaccc gtcgcctatc tgagcatagg aggaagtaag 840 tatggtctag ttatcgttga tgacttttcc cgcttcactt gggtgttctt tttgcaggat 900 aaatctgaaa cccaagggac cctcaagcgc ttcctcagga gatctcaaaa tgagtttgag 960 ctcaaggtga agaagataag gagcgacaac gggtccgagt tcaagaacct tcaagtggag 1020 gagttccttg aggaggaagg gatcaagcac gagttctccg ctccctacac accacagcaa 1080 aatggtgtgg tagagaggaa gaacatgacg ctaatcgata tggcgagaac gatgcttgga 1140 gaattcaaga cccccgagtg tttctggtcg gaagccgtga acacggcttg ccacgccatc 1200 aacagggtct accttcaccg cctcctcaag aagacgtcgt atgagcttct aaccggtaac 1260 aaacccaatg tatcttactt tcgtgtattt gggagcaaat gctacattct agtgaagaag 1320 ggtagaaatt ctaagtttgc tcccaaagct gtagaagggt ttttgttagg ttatgactca 1380 aatacaaagg cgtatagagt cttcaacaaa tcatcgggtt tggttgaaat ctctagcgac 1440 gttgtatttg atgagactaa tggctctcca agagagcaag ttgttgattg tgatgatgta 1500 gatgaagaag atgttccaac ggctgctata cgaaccatgg cgattggaga agtgcggcca 1560 caggaacaag atgaacgaga tcaaccttct tcctcaacaa cggtgcatcc cccaactcaa 1620 gacgatgaac aggttcatca acaggaggca tgtgatcaag gggagcacaa gatgatcatg 1680 tgatggagga agaagcgcaa ccggcacctc caacccaagt tcgagcgatg attcaaagga 1740 atcatcccgt cgatcaaatt ctgggtgata ttagcaaggg agtaactact cgatctcgat 1800 tagttaattt ttgtgagcat tactcctttg tctcttctat tgagcctttc agggtagaag 1860 aggccttgct agatccggac tgggtgttag ccatgcagga ggaactcaac aacttcaagc 1920 gcaatgaagt ttggacactg gtgcctcgtc ccaagcaaaa tgttgtggga accaagtggg 1980 tgttccgcaa caaacaggac gagcacgggg tggtgacgag gaacaaggct cgacttgtgg 2040 caaaaggtta tgcccaagtc gcaggtttgg actttgagga gacgtttgct cctgtggcta 2100 ggctagagtc aattcgcatc ttgctagcat atgccgctca ccactctttc aggttgtacc 2160 aaatggatgt gaagagcgct ttcctcaacg ggccgatcaa ggaagaggtg tacgtggagc 2220 aaccccctgg cttcgaggat gaacggtacc ccgaccacgt gtgtaagctc tctaaggcgc 2280 tctatggact taagcaagcc ccaagagcat ggtatgaatg ccttagagac tttttacttg 2340 ctaatgcttt caaggttggg aaagccgatc caactctgtt cactaagaca tgtgatggtg 2400 atttgtttgt gtgccaaatt tatgtcgacg acataatatt tggttctact aaccaaaagt 2460 cttgtgaaga gtttagcagg gtaatggcgc agaaattcga gatgtcaatg atgggcgagt 2520 tgaactactt ccttgggttc caagtgaagc aactcaagga tggcaccttc atctcccaaa 2580 cgaagtacac gcaagatcta ctaaagcggt ttgggatgaa ggacgccaag cccgcaaaga 2640 ctccgatggg gaccgacgga cacaccgacc tcaacaaagg aggtaagtcc gttgatcaaa 2700 aagcataccg gtcaatgata ggttctttac tttacttatg tgctagtaga ccggatatta 2760 tgcttagcgt atgcatgtgt gctagatttc aatccgatcc taaggagtgt cacttagtgg 2820 cggtgaagcg aattattaga tatttggttg ctacgccttg cttcgggctc tggtatccaa 2880 aggggtctac ctttgacctg gttggatact cagattccga ctatgttgga tgtaaggtcg 2940 ataggaagag tacatcgggg acgtgccaat tcttaggaag gtccctggtg tcgtggaact 3000 ctaagaaaca aacctccgtt gccctatcca ccgctgaggc cgagtatgtt gccgcaggac 3060 agtgttgcgc gcaactactt tggatgaggc aaaccctccg ggactttggc tacaatctga 3120 gcaaagtccc actcctatgt gacaatgaga gtgctatccg catggcggaa aatcctgttg 3180 aacacagccg cacaaagcac atagacatcc ggcatcactt tttgagagac caccagcaaa 3240 agggagatat cgaagtgttt catgttagca ccgagaacca gctagctgat atcgaagtgt 3300 ttcatgttag caccgagaac cttttgcagg ctgcgtagta agctaaatgt cttagattcg 3360 cggaacttgg attgaattgt agcatacatg tgtttatgcc tttgatcatg ttcattctgc 3420 attttgttgc ttattgtggt gctcaagttg tacaaacact ccctggatct cacaagtccg 3480 ttgcaaagtg atgctgagag cacctagagg gggggtgaat aggtgatcct gtaaaaactt 3540 51kk aacttatagc cacaaaaact tgttaaaggt tagcacaata attgccaagt ggctaaagag 3600 gagatcttgc acaatacgat tatcacagag aattcaacac agaggagaca tagtgattta 3660 tcccgtggtt cggccaagta caaaacttgc ctacttccac gttgtggcgt cccaacggac 3720 gagggttgca atcaaccctt ctcaagcggt ccaaagaccc acttgaatac cacggtgttt 3780 ttgccttgct tatctttatc ccacttacga ggaatctcca cagattggag tctctcgccc 3840 ttacacttaa gattcacaaa gaagcacgga gtaagggagg gaagcaacac acacaaatcc 3900 acagcgaaat gcgcacacac acggccaaga atcgagctca aagactatct cacagtttct 3960 cacaagaaca gagctcgaat cacttagaat cacaaacgga tgcgcaaaga ctgagtgtgg 4020 atgatcaaga atgctcaaag gttgcttggt gtctccctcc atgcgtctag gggtcccttt 4080 tatagcccca aggcagctag gagccgttga gaacaaatct ggaaggccat ctttgccttc 4140 tgtcgtcggg cgcaccggac agtccggtgc ccgattcctt tccttaattg gcgaagccga 4200 ccgttgcaga tgcgggagcc gttggcgcac cggacatgtc cggtgcacac cggacagtcc 4260 ggtgccccct tccgaccgtt ggctcggcca cgtgtcccgc gcagatcgcg cggtcgaccg 4320 ttggctcagc cgaccgttgg ctcaccggac agtccggtgc acaccggaca gtccggtgca 4380 caccggacag tccggtgaat tttagccgta cgccgtcagc aaattcccga gagcggcctc 4440 ttcggccaag gcagcctggc gcaccggaca ctgtccggtg caccaccgga cagtccggtg 4500 ccccagaccg aaacagcctc ttggctgtac acagccaagt cttctcttct cttcttcttt 4560 ctgtttctaa cacttagaca aatatattag tacacaaaac caatgtacta aggcttagaa 4620 acataccttt gctctagatt ttcactttgt tcatccatgg gcattgattc acatttaagc 4680 acttgtgttg acactcaatc accaaaatac ttagaaatgg cccaagggca catttccctt 4740 tcagatgcac agtttgaggg ggagatgtgt tacaacttga ccctttgaga ctaaccgtat 4800 ' gcttgagttt gcttgtttta gtctcaaagg agaattaaaa gggaaaaggt ggacttggac 4860 catgaaagac ttccactgca ctccgatgag agggtagctt attccaagtt catctcatgt 4920 actcttattg cctttgtatt cttattgaag attttggtga ggcaatgggg ttcttgggcc 4980 aagattgatc ctgttttggt gcttgatgcc aaagggggag aaaataaggg ccaaagcaat 5040 aaatggatca gctaccactt gagaaatttt gaaaacagta gaatagagct tttggtttgt 5100 caaatctctt ttgttgtctc ttttgtcaaa agttggcctc ttgtggggag aagtgttgat 5160 tatgggaaaa agggggagtt tttgaaatct ttcctttgga atgactctcc ttatgcttca 5220 acatgtgtgt ttgacttaga gatagagatt tgagtttgat ttgcaaaaac aaaccaagtg 5280 gtggcaaagg atgatccata tatgccaaat tgaatcaaaa taaatttgag tttttatttg 5340 aagtaatatt gcacttgttc tagttgcttt atgtagtgtt ggcataaatc accaaaaagg 5400 gggagattga aagggaaatg tgcccttggg ccatttctaa gtattttggt gattgagtgc 5460 caacacaagt gcttaaatgt gaattcatgt ttatggatga ataaagtgaa aatcaagagc 5520 aaaggtatgt ttctaagtct tagtacattg gttttgtgta ctaatatact tgtctaagta 5580 ttggaaacag gaagaaaaag aaaagaaaag agttggctgt gtacagccaa gaggctgttt 5640 cggtctgggg caccggactg tccggtggtg caccggacag tgtccggtgg tgcaccggac 5700 agtgtccggt gcgccaggct gcctcggccg aagtagccgc tctcgggaat tcgctgacgg 5760 cgtacgacta taattcaccg gactgtccgg tgtgcaccgg actgtccggt gtgcaccgga 5820 ctgtccggtg agccaacggt cggccgggcc aacggtcggc cgcgcgatct gcgcgtgaca 5880 cgtggccgag ccaacggcta gaagggggca ccggactgtc cggtgtgcac cggacatgtc 5940 cggtgcgcca acggctctct ggcgggcaac gggcggctgc gccattttag gaaggaaatc 6000 gggcaccgga cagtgtccgg tgtgcaccgg actgtccggt gcgcccgacg acagaaggca 6060 aggatggcct tccagatttg ttctcaacgg ctcctagctg tcttggggct ataaaaggga 6120 cccctaggcg catggaggag tacaccaagc attcctacaa cattcctaag caccaagaca 6180 tcgatctcac gcattcgttt cattgtgata gcatctagag ctcttgttga gttgcgaact 6240 ctttgagttg tgttgcgagc tcttgttgcg acttgtgtgc gtgttgttgc tctgatcttt 6300 tgaagtcttg tgtgcgttgc tcattccccc tttgctctgt gttctttgtg aacttcaatt 6360 gtaagggcga gaggctccaa gttgtggaga ttcctcgcaa acgggattga gaaaaaaagc 6420 aagcaaaaca ccgtggtatt caagtgggtc tttggaccgc ttgagagggg ttgattgcaa 6480 ccctcgtccg ttgggacgcc acaacatgga gtaggcaagc gttggtcttg gccgaaccac 6540 gggataaacc actgtgtcgt ctctgtgatt gatctcttgt ggtattgtgt tttgttgaga 6600 ctcctttcta gccacttggc atttattgtg ctaacactta acaagttttt gtggctataa 6660 gtttaagttt tacaggatca cctattcacc ccccccccct ctaggtgttc tcacctatgc 6720 acccgtagct aggcttgagt caattcgcat attattggcc tatgctactt accatggctt 6780 taagctttat caaatggacg tgaaaagtgc cttcctcaac ggaccaatca aggaagaggt 6840 ctatgttgag caacctcccg gctttgaaga cagtgagtat cctaaccatg tttataggct 6900 ctctaaggcg ctttatgggc tcaagcaagc cccaagagca tggtatgaat gccttagaga 6960 tttccttatc gctaatggct tcaaagtcgg caaggccgat cctactctat ccactaaaac 7020 tcttgacaat gatttgtttg tatgccaaat ttatgttgat gatatcatat ttgggtctac 7080 taacgaatct acttgtgagg aatttagtag gatcatgaca cagaaattcg agatgtctat 7140 gatgggggag ttgaaatatt tcttaggatt tcaagtgaag caactccaag agggcacctt 7200 cattagccaa acgaagtaca ctcaagacat tctaaacaag tttggaatga aggatgccaa 7260 gcccatcaaa acacccatgg gaacaaatgg gcatctcggc ctcgacacgg gaggtaagtc 7320 cgtggatcaa aaggtatacc ggtcgatgat tggttcattg ctttatttat gtgcatctcg 7380 accggacatt atgctttccg tatgcatgtg tgcaagattc caatccgacc ctaaggaatc 7440 ccaccttacg gccgtaaaac gaatcttgag atatttggct tatactccta agtttgggct 7500 ttggtaccct cggggatcca cttttgattt aattggttat tccgatgctg attgggcggg 7560 gtgtaagatt aataggaaga gcacatcggg gacttgccag ttcttgggaa gatccttggt 7620 gtcttgggct tcaaagaagc aaaattcggt tgctctttcc accgccgaag ccgagtacat 7680 tgccgcaggt cattgttgcg cgcaattgct ttggatgagg caaaccctgc gggactatgg 7740 ttacaaatta accaaagtcc ctttgctatg tgataatgag agtgcaatca aaatggccga 7800 caatcccgtc gagcatagcc gcactaagca catagccatt cggtatcatt ttcttaggga 7860 tcaccaacaa aagggagata tcgagatttc ttatattaac actaaagatc aattagccga 7920 tatctttacc aagcctcttg atgaacaaac ttttaccaaa cttaggcatg agctcaatat 7980 tcttgattcg cgcaattttt tttgctagat tgcacacgta gctcatttat atacctttga 8040 tcatatctct ttcatatgct atgactaatg tgtttttcaa gtccatttca caccaagtca 8100 taggtatatt gaaagggaat tggagttttc ggcgaagaca aaggcttcca ctccgtaact 8160 catcctttgc catcgctcca agaaaaggac cttgtctttg ggggagagag taaaagccca 8220 aagcaaaagg actggacttc gtctttggta taatcttaac tcatttactt atgaccaaag 8280 gggaagatag tacttctatg ggctctaatg attccgtttt tggcgattca tgccaaaggg 8340 ggagaaagta tgagcccaaa gcaaaaggac cgcaccacca ccaatttcaa aaacttagtg 8400 ctttctaaga gtatttatca attggtatcc tattgtgttc aaaaggagga gaaaattagt 8460 tttttcaaaa atgtatatca aaaccctctt gaacactaag aggtggatct cctttagggg 8520 gaattttttg ttaagtcaaa ggaaaagcat ttgaaacagg gggagaaaat ttcaaatctt 8580 gaaaatgctt tgcaaactct tattcattta cctttgacta tttgcaaaag atcttttgaa 8640 atggatttac aaaaagaatt tgcaaaaaca aaacatgtgg tgcaaacgtg gtccaaaatg 8700 ttaaataaga aagaaacaat ccatgcatat cttgtaagta gttatattgg ctcaattcca 8760 agcaaccttt acacttacat tatgcaaact agttcaatta tgcacttcta tatttgcttt 8820 ggtttgtgtt ggcatcaatc accaaaaagg gggagattga aagggaatta ggcttacacc 8880 tagttcctaa ataattttgg tggttgaatt gaccaacaca aataattgga ctaactagtt 8940 tgcccaagtg tatagattat acaggtgtaa aaggttcaca ctcagccaat aaaaagacca 9000 agttttggat tcaacaaagg agcaaagggg caaccgaagg cacccctggt ctggcgcacc 9060 ggactgtccg gtgtgccacc ggacatgtcc ggtgcaccag ggggactcag actcaaactc 9120 gccaccttcg ggaatttcca gaggcgactc ggctataatt caccggactg tccggtgtac 9180 accggacatg tccggtgctc caaggaaggt cggcctcagg aactcgccag cctcgggttt 9240 tctccctcgc cgctccgcta taattcaccg gactgtccgg tgtgcaccgg actgtccggt 9300 gcaacctcgg agcaacggct acttcgcgcc aacggctacc tgcaacggca tttaatgcgc 9360 gcgcagcacg cgcaggagtc agaatcgccc atgctggcac accggacagc aaacagtaca 9420 tgtccggtgt gcaccggaca cccaggtggg cccacaagtc agaagctcca acggtcagaa 9480 tccaacggca gtgatgacgt ggcagggggc accggactgt ccggtgtgca ccggactgtc 9540 cggtgcgcca tcgaacagac agcctcccaa cggccacttt tggtggttgg ggctataaat 9600 accccaacca ccccaccatt cattgcatcc aagttttcca cttctcaacc acttacaaga 9660 gctaggcatt caattctaga cacacccaaa gtgatcaaat cctctccaat tcaacacaaa 9720 gccctagtga ctagtgagag tgatttgccg tgttcatttg agctcttgcg cttggattgc 9780 tttctctctt tcattctttc ttgtgttcaa tactcacttg taaccaaggc aagagacacc 9840 aattgtgtgg tggtccttgc ggggaggttt ttctcccggt tgatttgaga agagaaagct 9900 cactcggtcg gagggaccgt ttgagagagg gaaagggttg aaaaagaccc ggcctttgtg 9960 gcctcctcaa cggggagtag gtttgagaga accgaacctc ggtaaaacaa atcctcgtgt 10020 ctcacttcat tatttgcttg cgatttgttt tcacgccctc tctcggactc gattatattt 10080 ctaacgctaa cccggcttgt agttgtgttt atatttgtaa atttcagttt cgccctattc 10140 accccccccc ctctaggcga ctatcaccag gcggtagtcc gcgagccaca gttccggtct 10200 cgtttccccc gaatacttcg tgatagtagt cgggggtcgg aaccgggtcg ggaacgacgc 10260 ccgtcggatg gcccgactga aggcttgcgg accgggtggt tcgggcgagg gactccgatc 10320 cctccccact gtcgtagcgc ccccccacgc ctggggtggt agcctcggcg caccctttcg 10380 tcgaggtggg cccgacggtc gcgtcgatgg tgctcgttgc cgaggtggcc cggggccgca 10440 ggcgcggtgt tgcgcgtgcg tccggtatag accgaggctt cccgcataaa ttgggaagtc 10500 51mm uutg 086ET be4q5qopo4 bbbgpoboe4 bo4boqbo4o q4qbpog4ob oboopbobob ooqbbbbqpe 0Z6ET ogppoopopb pobboqbPqb boebbgeqop bqbogobobo ogoqoobpog qbop000pbb 098E1 bob4poop5o bbeoeog446 b4qbb4006o ooPobo4o4e obpgebgpob poopoboopo 008ET pp000bboob bqqbsoqbbb qbeboqbsoq Poogqoqopo oo5qpoo5e4 ooqbpqopoo OD'LET goqp4op3o6 bboqbqopoo PPPPOPPPVP ebbPpmebPP PpPgupPbqo PPPPP4PPPe 089E1 544oepebqq Tep5p4;p4b qoppspoq4o epo4o4PPb4 4.4.4sbb.44Te o54bqqb.4.4q.
oz9T qbp5bge54p qoqPopPP4p EPP4PP4eP4 PP4e5P4.6PP p-44-454p4 p5eb4qppo5 09SET 040444P54b 4.646P4q44e 444PuebP4q pqpqbPbpbP 5ebPqqP4o; .64444;438s 00gET qbbe40-45PP 5Pqq44.644b bbepP0000P PPqP4DeP3P boopoqbbqp qquPq0000P
Of7t/ET 4bbbpbq4o4 bob5p4qesb pbqobpooPq 4bb555ge54 obTemobe obqoqoobbe HEE' o4p000pobo qppqqopbqo ooqpbqopbb Teqboqopoq bpboqPbqbo bppbobbboq OZEET obbp4obbpb oobb4q0005 qobbo455pb oqopo45pp5 popbpbobbb o400bbb6bb (1)9E-E epppgqboob ooqbobgebe bboobebobb boqoobbpPo obppbobpqq. oqgobqqbbb ()HET eQqbe444eb obb3b4bbo4 beogbbo4op p3b43o4ob4 pee44bobqq. 400poobbeo OD'TET qb45;Pep4p bbpoqbobob obbbbbboob 4o4obBoggo pogbbobbou bqbpbbobbo 080E1 b4bb4beogb 5ooe5poqb4 3qqopqb4ob obbobbpobo b54be5boqb po54opob5q OZOET bpboqb4.4.4o poqopelpoqb qoob4op5qo o5bepo55.44 4005o55qe; ooggobsbbo 096I bpbbobbboq bbbqopobsb 3o4be533o5 pb4obbeboo 44o4bo4boq qbpbbobubb 0Hz' obbBoqbbbb 4000bpbooq 5pb000bs44 oq5b5oog4o g5oobog45p 55o5p65obb Of78ZT bo4bbbbq3o obpbooqbeb opobpqqo45 b5oo4qog53 qbogqbebbo 5ub54bbbo4 08LZT bb5544qobe booqbpb000 5po4o4b55o ogq04b045; 445pbbo5ue bobbboqbbb OZLZT b4poobe5oo qbebbobbbp pobbebpqbb oqq.boqbbpb bobbpbobbb oqbbb4opoo 0993E beb000bebo obbeboobb4 boogq.o4b4o 4ppbo45bpb oobppbooqo oqobbbbbqb 009T opbobbboqb bpg000bqbq oob5bobbgb opbqq.b000; gobobbgebq pbqebbegbb Of7SZT bbo4ob534b eb000pb4bo q4o4gb4404 bboopoebbb ba5poobp4o 5ebbqbb4o4 08ZT P0005qqu04 poo5obb5b4 54qo5e5pop ogbo4boobo obp5e6o,o52 bubePqbooq OZZI 4054403004 5oP5406440 o45Pbo4b5o 5Pbbog54ob popbbbpbbo bb400bebbo 09EZT PPobebbubb o4b4obbg5o qbqpbgbobb qoPqopopob bqqoqPb-ebp bboobpo554 pout bqppoob4po eq5pp4000b obp4obubpb bbebbo5p4o 4bqboegob4 bp5eobeq54 Of7ZZI bbbbbTepop 4bg55poo4p bbebpbbepq bo5bpqe4gq ;o o55 g000bbeebp 08TzT p4oqo4400p poobbobobo poo4bogooq boqo45400b obpbpbepoo oobbobpbob OZTZT webbbebqbb bobopoogbo bppopqqbbb 555P4b2o5q 4o5ob45bbo 455p000bob 0901 000qboqqbq boqqoobbob 000qpbebbq bbubpbebqb obbboebpbb oogbgbbebo 000Z1 bbpbpbbbbb bppbobobpb obbbgbobob e5o3bbb4p5 p0005poopo b453530545 Of76TT 4bPbqbpbop boobsb0000 qbbbbbboop bpbombob bo4b3oppoo Pobobbqooe 08811 goopobbeob eobopobeo4 bo44bqobee b55oeb4gob pb54o4p000 b4oebeb54o OZ81T bqq.bbp5Pbb oobbb000qb ooPobebbou PP50004opb peoqopp54p oubbppboqb 09L11 oob335p4bo 4b4Po4p4bp bbeebbqbbo 5535050453 g5336-44poo 5oobbo4bqe OOLTT bbbobbboeb 005oo5oogq 5bo3boq54P bPPbbbbobo boopoo.4444 4544b4PPbo Of/911 405e6obb5b oebbpEcemb 5535544603 4334034035 0030544554 qoo5b44oeo 08S1T oo400bbo5o beebopboob p3P5o3o5og gebogbopbb bbgobobbbb bgboog000b cqgTT 35356=360 pboqbbpbob ouP5o3o45o 443o544obo bbopbbbbbb ob345453bp 09t711 04P5550g05 qq04403404 5055405450 5P5o5oo4-44 ob5so53oo4 boubobbbPb potTT opqb5eo334 Poqboopobq 3554opb55b 33Pop34bp3 boebobebbo boebogbbqq.
(3,E11 4qope3bp44 be 5e5553 p5pp4bop54 pepbo4bpbo obbpbogbbb obb4qobo4b 08TT bebobppeoo b000bobpbP bgepogoobo qopobqqbbb bqbp000pee pbopqpoboo OZZIT ebo5beep00 45504ee045 00480ebeb3 455e4b3035 bbebe300e6 534o34;o4e 09111 04503P5554 obqP5PoePo 000pppbebp opqbppoobq 4.555540p05 bgeob000gb OOTTT PP54poqb5o bgpopogbp4 bpobbpbebb o4bpopebbo 4bo3q4o5bo bqo5beeo4b of7oT1 oqbogooqbp oboobbbqqo bboboqgpoe pebbgbopbo 4o5pqopoo4 pbbbpbboqq.
0860T 00544.654e6 eeop.64-2056 004004500P 04peepb5p4 33.663opobb bpbobobbo4 0Z6O1 54P55bo53o o4obp4obpo PooPbbbboP ebbooboopo opebgbogbb b35g05-e504 09801 35433p5535 0003bo5430 bbgoobPbog 5340=4464 opoobobePo 43.45ogebbo 00801 H000qboeb 000b300p4e 000b000bbq qbbbpboobb 0030343.245 oPbgbbeobb 017LOT 554303e5e5 5305355053 P535.5q4504 e0P5400bP6 4305635506 55,054P5533 08901 bOODDP2DOP 53oq4bbpoo 5405505305 oq.Pobpbbpb bobgboqpob 50040553P5 onoT 3355355030 oopbooboq3 sbbg000gog 35p543o33p bpbbpooqqo obobpoboo5 09s01 5504500055 04340545e0 bbebbb0040 0530004e4b 5.55e500445 fiet4bobba5 caccacggaa cacagaagac agcaagggaa agaggaaggg caacttggag cagaagaaga 14040 agaatagggt acgcaacgtg gcggatgttc tttgttattt cgttcatatc ctcagcagcc 14100 tagcacatag tctatatatg tcactcctga actggactgc cacaatacgg cttacacggt 14160 ccactgcggc ccaaccacat ttaagtttgg cttcctggtc ctcagcacgc gaggctgcat 14220 catctcctga tgtgttgccg ctgtctccag gtcttgattc ccacttgctg ccagcttctt 14280 cttgtcttcc gacgacgctt tgctgacatt cccctgtcct cgagcgccag cttggtccca 14340 gatcaacgct tttggaaacc gagcacgcag tgcctcaacg tcctcccagg tagccagatc 14400 ctcagtcatg cctgaccaga ccaccttgac ctgcgcgatt aactcaccac cacggtcaat 14460 gaagcggcat tgtagaatac atgtaggcac ctggataggg ccaacatctt ggggcagctg 14520 aggtgaggcc cgttgatcac gcccgatgac ccgtttgagc tgtgacacat gaaaaattgg 14580 gtgaatagag ctggaggccg gtaaagctag acggtaagcc acggaaccca gacgatcgta 14640 atctggaaag gaccaaagaa tttgaaggac aacttgtgat tggagcgtgt agcaaccgaa 14700 gattggatat aaggttgtaa cttcaagtaa acccaatcac ccaccaaaaa cacgcgttca 14760 ctgcgctgct tgtcagcttg ccgtttcata cggtcttgag ctcgatgcag atgcaattta 14820 atgactttgg tcataagctc tcgttccttc aaccaagttt cgagctctgg ctgtggcacc 14880 acaatatcaa ccaaaattcc aaaatatcgc ggagtgtggc catataacac ttcaaagggt 14940 gtacgcccca aggttgaatg tactgtggta ttgtaccagt attcagcaac tgacagccaa 15000 gctgaccaac gtgaaggaca agtgtgcaca tagcaacgaa gaaaagtttc caagcattga 15060 ttcaagcgct ctgtttgtcc atcggattgg ggatggtaag aagaggacat ggacagattg 15120 gtacccgtga tctgaaatag ttgttgccag aatgagctag taaaaattct gtcgcgatct 15180 gaaatgatgt tgacaggtaa cccgtgtagc ttgtacacat tgtcaagaaa aacacgagcc 15240 actttagcag ctgtgaaggg atggagtaaa ggtaagaagt gtccatactt cgaaaattta 15300 tccacaacca ccaagatgca gttatagtga gctgaacggg gcaaaccttc aataaaatcc 15360 aatgaaactg tatgccacgc ttgtggtgga acttccaaag gctgcaatag gccaggatat 15420 ttagcacgat cgggcttgga ttgctggcat accgtacaag attgaacgta ctgtagcaca 15480 tcagcacaca tacctggcca atagaacatt tgtttcactt tgtgatatgt tgctggggct 15540 ccagaatgac ctccgaccgc cgtatcatgc atagcttgta ataccttctg ttgcagctgc 15600 aaattgccgc ccacccagat tctatttttg tgtcgaatga ttccttgatg caaggaaaaa 15660 ggagctttgt ctgcagaatt caaaattaat tgagccagca actgtttact agtaggatcc 15720 tgtcatagc cttccactcc ctcctgcagc caagtaggca cactgtgaga aatagcacaa 15780 cagtgactat cttcctggac ttttcgagac agtgcatctg cagctccatt atccacccct 15840 tttctgtaga caatcttata ttgcaagcca gccaatttcg tatacatttt ctgttgccag 15900 atagtatgta aacgctgctc attcagttgt gctaaactcc tgtgatctgt atagataatg 15960 aactcagcca actgaagata ggacctccat tgagcaatgg ctaaaataat ggccatgtat 16020 tccttttcat aggtcgagag tccctgattt ttcggtccaa gagccttgct cagaaacgct 16080 aaaggatgtc cactttgcat aagcaccgca cccacaccat aataggaagc ataagtatga 16140 atataaaatg gctgggaaaa tcaggcagag ctaacactgg tgcagtgacc aatgcttgtt 16200 tcaaaacttc aaaagcttta aaatgatcca ctgtccacac aaataaagtg tgtttcttca 16260 agagatcaaa taaaggtcta ctgataattc caaagtgctt gacaaatttt ctgtaaaaac 16320 cggctagacc caggaaacat cgcagttcct tagcattggt tggtactgcc caagaggata 16380 tggcttgaat tttactagga tctgtggata ccccttgctc actgatgata tggcccaagt 16440 aagcaatatt tgtttgagca aattcacact tagataattt gactttccaa ttatcagaca 16500 acaataattg caaaaccttc tgcagatgca gtagatgctc ttcaaatgac ttactgtaaa 16560 ccaagatgtc atcaaaaaaa ccagagcaca ttttctcaat actggtttca gggtttcatt 16620 cgttgcactt aagaatgtgt taggagcacc tgtcaagtca aaagccatca ctctaaactc 16680 atagtggccc acatgagttt gaaatgctgt tttatattct tcgccagatt tcaaaagaat 16740 ttggtgatat ccagctctga gatccagttt actaaaccat ttagagtggg cgagctcatc 16800 aatcaattgg tcaaacacag gaactgggta tttgctcttg agagtgaggg cattcaaata 16860 cctatagtcc acacaaaatc gccatgtcat gtcttttttc ttgaccaaca ccatagggga 16920 agcaaaagaa ctattgcttt tctgaataac accttgatga aacatctctt gaacttgttt 16980 ttcaatttca tccttcaggg ctggaggata cctgtaaggt ctgacagaca ctggctgagc 17040 accttccacc aaaggaatag catggtcaca atccctgctt gggggcaaac cctgaggttc 17100 gtcaaagact gagctgaact gttgtagcaa tgcttgaatt gcaggatggc tgtcaggaga 17160 agagcctacg gaactgacag attccatgaa caacaactct atcatagtat cagcaggtac 17220 ccctggggtg ttgccctgca gcagcacaga agagccttga tatggtataa ttagccactt 17280 ttgagcccaa tccactctca tgggactaaa ggatttaagc caatccatgc ctaccaccat 17340 gtcgtagtag ggaaggggca ggaatgaaac gtccgaagta aaactgcagt tctgaatctg 17400 ccattgtgcc tgcaacaatt tgtaatgaca agtcaccata gccccattgg ccacttgcac 17460 5loo ttgcagagta gatgccatag atgtcacccc ttgcaagtgt ggtctaagtt gatcattcaa 17520 aaaggtgtgt gagctgccac tatctatcag aataagtaag ggatgattct gaatgctccc 17580 atttaatttc agagtttgtc gaccagtcga ccccgtccat gcagatttgg aaatagtcac 17640 gaacaactgt tctagagcag gttcaggtgg agataagtca gattcgggta cttcttcatc 17700 caccaataat gaccaaacct cctccatagc atgaagttga gcagtggcaa cacatttgtg 17760 accgggattc cacttttctg cacacttgtc acaaagaccc tttgctcgac gaaaacgtcg 17820 caacgattcc agcttatcag aatgagatgc agattgagtt gaatccttgt ttgaagtcca 17880 tttagttgaa gcagacaact gcacaccagt cttggggcca gcatgacttg aatatggttc 17940 agaacggcgc caacgtctcg ccgttgtggc ctcctcctgc accaacgcga gagaacaggc 18000 agtgtccaaa ttcgacgggc gttgcaccat aataacagct ttaatatcat cacgcaaacc 18060 atctatgaac cgcattgtgt aatacaatgg gtcagcattt gcttcatatg cagacaagtg 18120 atcaacaagt attgaaaatt gttctacata ctcagctacg ataccggact gatgtatgtg 18180 gaaaagttga cgaattaagg attcatgctg ctctctacca aatctatcat gaagctggcg 18240 acaaaattca gaccacgaca acatacgcac cctctgacca acagactgta accatgatgc 18300 agcacgccca ataaaatgca tagtagctac acgaatccac atatatggtt ccacgtcata 18360 catatcaaag taattttcac aaagcgtctt ccacaactga ggattgtccc cgtcaaagtg 18420 agggaaatta accctcggta ggttaccgtg cccacagcga aacccctcgt gaccgccatt 18480 cagttcagtg tgacgaacag aatcatcaaa tggatcaata cgaggtgaat cgtggtacgt 18540 acccttgacc gggacatggg tttgagcatg accatgccca aacccacgcg cccggtgaca 18600 tggttcagcg tggtggccaa atggcccgtc agcgggttgt ttcccggcag atggatgctc 18660 ggacgccaac ccggaaggag tgaagagacc tggcttggtc tgatcagcgg cgatcgtctc 18720 acgctccatg aacttggtgg cacgcttgct ctcgaggaag aggttctcca gacgcctctc 18780 cacttctggg cgccaagtat ccacctcgga atgccccatc ttgagtgaga tgaagcgtgc 18840 ctccgcttgt tgcgccatcg cgtcgatccg ttgctcaatc gcgccgcagc gattctccag 18900 cgtcgcagcc atgcgctctt ccatctgcga caaggcctct agaaccttct gcatcgcgga 18960 atccataccg gcgaccgcag tggatcgagg gcgccgaccg ggtatgggat ccagattctc 19020 taggatgaac tagtctgata ccacctgtta gcaccacgga acacagaaga cagtaaggga 19080 aggaggaagg gcaacttgga gcagaagaag aagaataggg tacgcaacgt ggcggctgtt 19140 ctttgttatt tcgttcatat cctcagcagc ctagcacata gtctatatat gtcactcctg 19200 aactggactg ccacaatacg gcttacacgg cccactgcgg cccaaccaca tttaagtttg 19260 gcttcctggt cctcagcacg cgaggttgca tcgtctcctg atgtgttgct gctatctcca 19320 ggtcttgatt cccacttgct gccagcttct tcttgtcttc cgacgacgct ctgctgacag 19380 tcaccgcccg acagacttgt gggcccgctt cgccagaacc ttcgtctccc tcccgtaacg 19440 aactagggca caacacaaac ttgtacttgt gtagccgggg tgtgtttact ggccgaccgc 19500 acccgccttg gactcgtcca ctgcccctat ataaacggtc gccccccgcc ctcgatcaat 19560 cagagtttag gagcccctgc tacctgttgt cgtgtggcgc cgtcgcaatg agctcgacgc 19620 cgcacgccat cgtaattcag ccacacctac gcttttgcct tgctttcggt aggaggtttg 19680 agggtttcgc cgtcgtacgt gggagctgct ggatgtttcg ttaggtgagg gaatctactg 19740 gaggcaggca ctgcgtgccg aggatcaccg ttgcatccca agccaccgtt cgacgtcgcc 19800 gactctcgcc ctcaatttag ttaccgacga ggaccccttg actgtttcgt ttgcttctca 19860 ctcgtaccta ggcacgagta gcgctttaga tcggttttgg ggcactcgga ttgctcgccg 19920 gtgttcagag attaccacag agtcacgctg tgctgagcgc gaggctcgac gctgcatgat 19980 cattgatcag accttgtccg tgttgtcttc attgaccaca gcttcgcata ccaccatttg 20040 gattggagaa atagagcgca aggtcgtcgg ttcgcgtgtg ctagcgagct gtcagggcgg 20100 agctctattt ctgccaccat gacgcgttgg tagagaaagg agcggcatca gttgatcagg 20160 attaaatccc gcgtacccct tcagcacatt aaatcaaagc cgtcagatct aatctagacg 20220 tttgtgattc gataatagcg tgtcggttta gtatttaaat ctggaccgtt gatctctaga 20280 tgatcgcctt aggtcgtgta ccagttagtg tagttgggtg aactttatta aagagcccct 20340 ataaaattta ggaattaacc tgcaatcacg tgatatttag aaagtcatgt agctaggtca 20400 tgttcttaac atattagacc cgatggattt tagaatcgga agtacacatt aaaggtatga 20460 ttctatactt agtacattag tttttgtgta ctaacacatt tgtctaagtg ctagaatgag 20520 aaaaaagaca aaaggaaaaa gagttggctg tgtacagcca actgctgttc agtctgggtg 20580 caccggactg tccggtgagc ctacagtcgg ccgcgcaatc tgcgcgtgac gcgtggccgg 20640 gccaacggtc tgatgggggc accggagtgt ccggtgtgca ccagacagtg tccggtgcgc 20700 caacggctcc aaatcttcaa cggtcggctg cgccaaaata ggaaagcaat cagcaccggg 20760 cagtgtccgg tggtgcaccg gactgtccgg tgcaccaccc gacagaaggc aaggataacc 20820 ttcctggatt gctctcaatg gctcctagct gccttggggc tataaaaggg acccctaggc 20880 gcatagagga gtacaccaag catactataa gcattcttga tcattcacac tccgtctctg 20940 lpp ctcactcgat tgactttctt agtgatttga gctccgttct tgtggcgaat cttgtgctat 21000 tcatttgagc tcaagtcttg gctgtgtgtg cgtattgctg tggatttgtg tgtgttgctt 21060 ccctccccta ctctagtgct ttcactttga tccttattgt aagagcgaga gactccaagt 21120 tgtggagtaa atgaaactga accctaaacc ctaaaccctc taaaaatgac taaaatgcaa 21180 aatagacggc gcatacataa ctaggagtaa aatgtccgaa aaaaaatcgg ctcgagtctc 21240 gagactcgag tgccctctct gcctataaat cgaaccctaa ccctttttcg tacctgtttg 21300 tgtccttagg gtttagggtt ctctgctcat tcgttcgcca cctcgcccca agacgtctcg 21360 ctagggtttc gccacagccg ccgccatgcc tcgccgcaag cttaggtaac cttctcctct 21420 ggcgcctcct agggttttgt ttcgagattc ggttgttcct tgcgttgacg ggccggggct 21480 ggctcctcct ggatctgggg ctcaggcgcg taggcttggg cgttagtaga tttgattcag 21540 tcggtatgat gtcgttaatc gtttgtttta gtatgtgcaa atgatactgg ggttgtgggg 21600 ataggattgc gcatgtttgc catgtttagt tgcagaaata tccggatctg tttcttgcgt 21660 tcaatgggct gggtctcttc ctgtttagat aattgtaaga aatggacggg tgttcataat 21720 cgacagagat ccgattgctt ctcgaataac cgattgcaga tactatctgt tcgcaggcgg 21780 ttcagacagg ggcatagaac aaatctgaat ccccacgagg gaaggtttat ttccattaat 21840 cctttctcgt taggattcgt atagatttac atatatacta caggttgtct tatgaggtgt 21900 ttggtatatt cttgaaagtt aattcaccag ggtagtggac tgaatcttat gaatgttgta 21960 tgtgtgtagc tgtattgtat tgtccgttta taatgcctct ttgagtagcc attacagtcc 22020 ctgagtactg tcttggttta gttagggggc gcaaagcact ggacatctga tgtccactca 22080 ggccttttga gggggtacct cacctgtctt accaagaagt gccccccaag cagccttaga 22140 catacatcta cataatctct gtttgtaagt gcctctttga gtagccatta cagtccctgg 22200 gtactgtctt ggtttagtta gggggcacaa agcaatgggc atctgatgtc cactcacctt 22260 ttgaggggca ccttatctgt cttacaaaga agtgcccctc aagcagccct agacatatat 22320 ctacattatc actgtttgat ctgtcctcaa tgccacgtct tttcacttag tttttggcac 22380 tcatatttgc tttattgagt tgccactgta gttgtatata caatcttggt ttagttagga 22440 ggcacatgtg atcttgaaaa aaactgacat tgtagtgtta tcatctttcc tgttgaatgg 22500 tagacatgta atgcaaaaca ttcaaaagat tgcttgtgta cttgattagt atcaaggttt 22560 cagggagcga tgacatttct taatatattt ggaggactca acttagttac taactggact 22620 acaagtaaca aataatgtgc ctcttgtctt tattcatccc ttcttagatt gattcctaca 22680 gttaactgct attttcatca tttttgctgg tggaagatgt ggttgagctt gctttacagt 22740 attttggttt tgttagcttg tgttgcatct ctctctctac atttattatg tgttgatttt 22800 tgagttaaaa ctttgtttat gataaaccat gttgctttct ttggttaatt ttttttgctt 22860 aatgcttatt cccccactgt actgtcagga aggtccgcgg ctcacgcccg gctccacatg 22920 ctgctccagt gaggaaccca ccacagcctg gtaaagtgat tcacgcagac aataagtatc 22980 tttagaactg acattggcat ggtaatcatg cctcttttga ctctgcagta ttctattgtg 23040 gacatgtgtt tcaattatgt aactttagtt atatttttgt ataactgttt gctcagttgt 23100 tgagaatgtg ttcggtttgt tactataaca atgttgatga cataaatata ctgttaagtt 23160 tatattggaa tttgtggtac aagtctgaag ttgttgattg tgtttgaagc agagtcaatg 23220 gcatttcggt ttatatagag aattacagtt ccttgttttt tggtagaact ggaacctgtt 23280 tcagttctgt tcctaatgct tacacatggg tttgtgctac aagtatagta ttagtattac 23340 agagctcatt gttttggaat cttgattgct ttggaatcag aatcgtttat gtttagctca 23400 tattagtatt agtattacag ctcattgtgt ttatatagga gttttaatct gttctatttc 23460 tgttcctaag tgtcttctga cttttctttt tggcagcgcg ctaggctcgt cctccagctc 23520 ctgttcgtga tggtggtggt ggctccattc ttggaggaat tgggtccacc attgcttaag 23580 gtagttttca aagcttgttc ttttttgaat aacttcagca acaaacatta tcataatcct 23640 tgaattgatt tgaacgaaca cattgtttta ggtatgccat ttggtacgag tagtgccatg 23700 gcacacaggg ctgttgatgc tgtaatgggt ctccggactg ttcggcatga gattgttatc 23760 tcagaagctg ctgctgctgc ccctcctgct ccagtgatga acgctaatgc ttgcagcatc 23820 cattctaagg ctttccaaga tgtatgtctg cttgccacct ttatgctgcc ttgttttccc 23880 tcaatttgat gcatgaaaca tttggttact cacttctgtg atgtagtagt gaagttttat 23940 ggtgtctttt tttttccttt actgaacctg caaattactt aatcatcaaa ccttggccat 24000 caatcaagtt ttaatattat gggcatgatc ctaccgttgg ctttgttgag gtcatgttaa 24060 gaattgttaa cctgcatttt gtatactcat tcgatgatgt gttctcaccg attttcttgg 24120 ttgatgcagt gtcttaacaa ctatggcagt gagatcagca agtgccagtt ctaccttgac 24180 atgttgaacg agtgccgccg tggtgttgtc tgcctgagct tttgctccaa ttgggattat 24240 tcatggattc tcttttcact cccatgtatc tcattaataa gacaccgtga aacttttaac 24300 cctctccacg aatgcaccat ggcatcacgg gttcatcttg ttgaatctgt ggttacgttc 24360 tctttatcct gtgttgttgg aacagacttc tgtctcttct ggtgatcata aatatttaaa 24420 51qq tgaaccagtt gtgttggaaa atgttgtttt cttttgtctc tagactggaa agcggagttc 24480 tcgtcaacac ggttctttca actagggatg aaagtggtaa tccgaattgt tagtacaaat 24540 ttaatatttt aaaatagata tgtataaaat tttatgttga tcttttttat gttatcaagc 24600 acattagtat aaattagtat aaatatgaat aaaatattac ataaaatgtt ttatgtatta 24660 tttggtccct acaacataaa tagttgaaaa aattactaaa tttgttttcg aatctatatc 24720 gaagtttata tctattattt aagaaaaata taggatgaaa aggtttatct tttatgaatc 24780 tttacaagct ggatcttata aacaagaaaa taaatttata ttgtagattt tatatcctat 24840 ttattcgcaa tcaaagaaaa gcgactaaaa aactgattac cgagtaaata ctgtttccaa 24900 ccgttttcgt ccctactatc aacgccttct cccaaccgca gtcgatctgt ccgtctgtat 24960 caggcgcagc ggcacccctg ctgttcgact atctagacca tagaatattt taggtataca 25020 ataattttag ttccacgcta gaacatttta gttagaataa taacaagatt tgctattgat 25080 gtaggactcg cccgtcactg tctaaaaaag cattctgtcg gtcttattct ttaggcatca .25140 gcgggtgtac tatctcattt ttcctatcat attcctcagt actctgttaa gtataaatgg 25200 tctattttac atgatgaact aataaaacta attaaggatc ctaacttttt gtgaaggtaa 25260 tttggatcat tatgcattac catcctacgt atacctgctg cagcagcatc tgcgtaagca 25320 cagcctagat atatgcttct gtgtggactg aaaggagact ttgtttatca attagtatac 25380 tcccaaaaaa ctgatgacac caatgatgca aataggctgg gaatagtctg tctaatagtt 25440 tgagtgaatc atgtcactgt gcgtcctctg caggcagttg ttgacatgag cgcatcgtca 25500 ctgctgaatc gccatggtct gaaggcaaca gataaggcat actgggcctt gtggtagttg 25560 ttttactggg cctttttgta tgatctataa aattcactgg gatcaacccg gagaggaatg 25620 gcagcagatg cagtccccag ggtcctccgt cgccgcctga gcacccggca cccgcgctga 25680 accggagagg gacgcgcgga cgccgtgcag ctggtgcgga gggggctgtg gcagatgagg 25740 atgagacgcg tacgtggctg ggaaggccag caggccaccg ggtcttcgtc cagcccggcg 25800 cgagtggaca ggactagaga tggcaacggt tacaaacccg ctgggtttta ccgtcccaaa 25860 cccgtacccg tgaaaaatat ctatgcccat taaaaaaccc gtacccatga cgggtttgag 25920 attttgccca aacccgtacc catcgggtta acgggtaccc atgggttacc cgcgggtttc 25980 atctccaata tacctgttct tctcataatc aataagtatc gtaatgatta atgatatcat 26040 gatccaaaat ctatgtaatg aacaacgagt tcatgatttg gtataaaaat tattagtaga 26100 gagaatgaaa tacaaataat aagttgtata attaagtgac cttgcactaa gttatccatc 26160 catcacatat ataacgctag taaaaactat aatatcaagc aagcaacact ctcaccgact 26220 actgatacat tcaccaattg ataaaaaata tgaagtaaat aaggaataac aagtttgttg 26280 ttcgtttata aaataaaatg acaatatgca ctaggtttgg tcgggtttaa aaaacccacg 26340 ggttcacggg tttgggtact ataggaacaa acccgtaccc ataaacccat tgggtacaga 26400 tttatgcccg ttaacaaacc catgggtatg aaaattgacc caaacctata ccctaatggg 26460 gtaaaaaccc atcgggtttc ggatttcggg tacccattgc catctctaga caggacaacc 26520 tcggccggtc ctgtatgtag gccaccagca tcggccagtt ggtacatcca gccggggtca 26580 ggtcactttt actcgtctca atcagacaat caccgtccac caacgaacgc caacgttgtc 26640 acttgtcagg tcggttgaga cttgtatttt tttttgtcct ccgtaaaaat cagttccaac 26700 gacagatacc ccgacggtaa gcggacagcg ctgtcgtagt cgttttgttt gagtccacaa 26760 atgagccgaa gatgcaagca gcatatgcaa cgtgtgttac aaagaaaaga agtggatgca 26820 aaggcactac gagaatgaac tggtgccacg gaaagatgga accaaaccaa cgaactattt 26880 tttccctcct tttttttatt aatgcttcgg acgacgatag cagtttctgt acaactcttg 26940 atatataaaa aacaaacaat atgacggatt aacctaggca tccaatgccc ccccaatttc 27000 ctcctgctat agcgccacac cttgctgctg cgacgactgg agccttccct tgaggaagcc 27060 cctgcacacc ttgctccaca gctccttgtc cggctgccgc acgtcgaagc tcgtcttggc 27120 cacttccacc tggcagtcct gccaacaaca aacaaaacaa ggacagatat ttttttttgt 27180 cctccgtaaa aatcagctcc aacgacagat accccgacgg taagcggaca gcgctgtcgt 27240 agtcgtttag tttgagtcca caaatgagcc gaagatgaaa gcagcatatg caaggtgtgt 27300 taaaaagaaa agaagtggat gcaaaggcac tacgagaatg aactggtgcc acggaaagat 27360 ggaaccaaac caacgaacaa ttttttccct cctttttttt tattaatgct tcggacgaag 27420 atagcagttt ctgtacaact cttgagttct gaagcacata taataaaaaa caaacaatat 27480 gacggattaa cctaggcatc caatgccccc caatttcctc ctgctatagc gccacacctt 27540 gctgctgcga cgactggagc cttcccttga ggaagcccct gcacaccttg ctccacagct 27600 ccttgtccgg ctgccgcacg tcgaagctcg tcttggccac ttccacctgg cagtcctgcc 27660 aacaacaaac aaaacaagga cagatatttt ttttccatgt cacagacaga cagacagaca 27720 gacagacaga gagacaaacg aacactcggc acgctgtcgt tactattatg attaattacc 27780 agtatgtggt tgtggcggag gaggcggtcg gcgatgctca tgaggacgtc cttgctgtac 27840 tccgggtggc cctggacgcc catggcgcgg ccgccgaggc ggaacatctc gacgcgggtc 27900 51rr ttgtcggacc gcgccagcgc ctcggcgccg gggggcagct cccacacctc gtcctggtgg 27960 aactcgatca ccggcatgtg cacgggcagc ttcagcggcg cgaacagccg cgccgccgcc 28020 gccgtcgggt ggatgcagct caccccgatg tcccagccct tggccgaccg ccccgtgcgc 28080 ccgcccagcg cccggcacag gatctgcgtg ccgcaatcaa acagcttcag gagttaagac 28140 ggaagtgtag tagccacagg agtttagagg tacgtgtgcg taacgtggac gcgtgcaagt 28200 ctgcctggca tcggcgcccc accaccgaca gggccgatgg acgcaactaa aacgttttct 28260 tcgggaacac gccaagtaat tgattcatgt gcacacagac tgtccccctt ctgtttgtag 28320 tacgatattg ggaacctcct aggattccac tgctaaacaa ttggtgtctc ctgttcctgc 28380 gtacgagtac gacgacggaa gacttcgcgg ccacaagagc aatccaaatc caaggctctg 28440 gctctcggac cacagggaca gcgacagtgt aagagctctt tgaagttgcg agtgttatta 28500 acatagaacc aagtaataat taggagaaga ggtcagtacg tcctcacgcg tcgaggcacg 28560 tctctgacaa aagcgtggtt cgcgcgggcg attgcgtgcg cttgggcttg ggcgctaccc 28620 gcacgccctt gggcgtgatc tgatcccctg agctgctggg ttggtacgct agtagcatcg 28680 tcatcccaaa agcaatctcg ctcggcaggc gagatgcgtc acgccatttg cgttcttcgt 28740 cctcctcctc cttcccgggg gatttgattt taaaatttgc aggcgcggtt tacgcgccac 28800 tttgaggcaa acaaaccggc gaaccggaat aggggaaccg ctcgctcctc taagccaggg 28860 caggggcgca gggctctgtg actgtgagcc aaacgtccgg ccacagacgc agcgggccgt 28920 aggagatcct gcagaacaga tacccactcc acttgtctgt gtgccaacaa caacagcaac 28980 ccagctgcca cggccacgtc acaccacgtg aatcagcgat gtcagcctcg agccttaacc 29040 ctttgtttga gagatgctgc cgtgtcttaa acctagatta gcgtggagtt tgtagtcact 29100 aatccatcca atcagtcccg tgtccgtctg tttgcttcga cccgaatcaa accaggggcc 29160 tctgcttcta tgaactgaac tggcatggac tgaaacaaaa cagcgtcatg gcagaagcaa 29220 gagctctata ttgtgcgctc taaatggaga ttggtaaaac tcgtagaatt gggggttcgt 29280 ttcttccgtc aaactctaac tgtgggcttg atgctagcac tgagtcacct ttgtcattct 29340 ctggggaaaa aaaataaggc atctttccgt ccccattgct actgctcacc aattaagcag 29400 ccagtaccgt ttgtactagt caaaatgcat caagaacgac cacgggtcgc cgtaaaaaaa 29460 aagaaagaaa ataatagaaa agaaaaggtg cgccgggtgg acgggaaggc gctaggctag 29520 aagagcggca gccccaccag gtggtgcccg cagcgtcccg cggtcccgtg attcaccagc 29580 gacgacggag aaacccagct acgcgcggaa gggactagac gagaaccaac cgcaccgcac 29640 ctaaacccaa ccaaacagca agtcgaacca aacatggacg gatgggatgg gcacctggtg 29700 gccgaagcag acgccgagga cgcgcttgcc ggcggcgtgg acgcggcggg tgaggtcgac 29760 gagcgcgagg atccagggct cgtcgccgtg cgcgtcggcg cagctcccgg agatgacgaa 29820 cccgtcgaac ccggctgcct cggcgtcggc ggggagctcc ccgcggacgg cgctgtacac 29880 ccgccacctc tcgccgtcct cctccagcag cgcgcggaag acggcgaagt agccgccgta 29940 cttctgccgc acgtactccg agtcctcgcc gcactgcagc accgcgtacg agcccgcccg 30000 cgagggggcg gcggcggcgg cggcggcggc ctccagcgcg gccgacgcgg cgttgtcgag 30060 cggccccatt cccatggctg gcggcgtcgc ggccaggcag gcgcgtctcc ggcccgggaa 30120 tggaggcggg ggttgggcgt cgcttggctt ggctgactcg cttgcggggg gtgagaggtg 30180 gcggggagga tggggggaga acggcgaggc gaggcgaggg cgatatttaa agtaagcacg 30240 agcggttttc cctcggattt tttttatttt ttcgtcgctg gttttccatt ttgctattat 30300 agtactgttt ctgttttttt ttactggtac tggggctggc cgctaccggc ctaccgctaa 30360 catttggtat ggacgtatgg catggcaacg gccagcaggc gcgggacggg ggctaccggc 30420 gagcggcggt tttgctcggg tccccttcac tgctcgggtc tttggccggg tcctagtaat 30480 gggtacccaa aatcgggtat ccgatagata aaaatcctat tagagcatgg atgtagcgaa 30540 ttttaatatc tatggatatt ttattaggtc atttattaaa tttcactcgc tcatgcatac 30600 aatatactta acaagcaaag aagacaaacg tggacccctt aatctcacct ttaaaacata 30660 tattgataaa agagtttttt tttctagacc gatcttgtca aatactgtat ctacctgcct 30720 gataaaagat tcaaacagga agagcaaaag cgtatatcaa attaattgac acgggagtcc 30780 atttctgtgc tcagagtaag cgagccctac aattgcaaaa aaaagggggg gggggggggt 30840 atgcatatgt aatatctgat gtcatttata tatagtgcaa cgattctctt tcgtaccaag 30900 ttgcaagtcc tcatatcgaa tcgtaagtta ttctgttttc ctgtgtacat atgaacatat 30960 ctatctttta atacaaacag cattttttat agttttttct aagtacatat gaacatatat 31020 ctatcttcta atacaaacaa catatttttt gtatagtttt tgttatgatc atatgttttc 31080 ccatgctacc gtttgtatta aaacacggaa actaaactac actaatttat tttattcata 31140 tttctttctt taaaaaaatg ttttttatcg ttccatcctc tctatttcaa accgtaagct 31200 gttctggctt tttttttcta aatgcgtatt ttagttatat ttctagatat aattagagtg 31260 tacataaaca cataaaaacc cacttactaa aatcacaaca acttacagca atttaaaacc 31320 gtgagattgc cgagcagggc gagaaccgac tgttagacat gctcacatgc atgtctgggg 31380 51ss cattgtttga ttttatttgc ccgagaaatt gctctgtttt cattcgtaga atttgtacta 31440 gtattttctt accgaggctg gctgggcata cgtgcacgcg cgcgatcgtg cagcgacatg 31500 cacatgcaat gcaatcaccg cgtgacggag taccgaggcg aggtcgtcga ccacgtgccg 31560 tatgggccgc tggcacgtga caccaccgat tcggatcctt atttattcat tcatttgtca 31620 gtccccacgc attgtattta aaaattcaga aacgtaagcc cacttttacg caaaaacaac 31680 tttactgtcc gaaacgcctt gtgcaactag ctaggctagg aggctaactc attagtaaaa 31740 tgttgtgttt tccacccttc ctttgtacta attttataga ctattagccc ggacgagctg 31800 gctaggccag acagggaaat tatattttac tttgacgcct catctggatc attggaattg 31860 aattccattc taacaatatt aatttaggta tatatcaatt aagctaatcc ggttttatgc 31920 aaaatatatt tatatactat tattagcaag atgtcggaga tatttatgtg ctacattttt 31980 actataaagg agtgaaacga agagtgtcat gtaaattaca gactagaaac gaattctact 32040 aatgcataaa atcatttcac acactccacc ccatgaattt gagatagcct tatatctgaa 32100 ctttggaaag tggtggaatg tcaaattcca aactaaataa gttattttat tgagtgaatt 32160 ccaattcctc taaaatgaag ggatccaaat gccccgtgac tggaatttgg agacgaagcg 32220 cgcgcagaat attcgggtca aatagaatca gctacatact atgtgcatta gggctcacat 32280 aaataatcca tatcttgagg agtatcaaga gagtaagtgt aaaaaaaata caagagacat 32340 atcttgatga agagatgtat ctagctcagt tctctaagtc tagatacagt ttcgtccata 32400 caacccacat aaatgagtta tatcatgaga gattctagtg aataagatat atgattatat 32460 acaaaaatag tttcttatat gtttttttgt attttgaaaa ccgaactgga gtcttaaggt 32520 tgcacatgcc ctggatgtat ctagtgtttg taaaacctgt ttgtaaaacc gcaacaaagt 32580 ttctacattg tacatgccct tagggatgtt accaattttg gtagtgaaac gcaataatga 32640 attaagcaat aattcaagcg gatcagaatt aaatgagacg tgtggtgtag actgtagaga 32700 gtactctacg atatgtagtg ggatacttgg agcagtaatt attactaaac atgaggtgta 32760 gtaaacattg acgggataac gctgcacatt aaatactagt atcagtaaac gatgcaatat 32820 gcacgatcat ggtccgagag aatattcgga tcaacacatc tatctcctgc gtttatatat 32880 tattaaaact gaacgttacc gttttttgtt agtagtgata aagatcgatt aaggtaactc 32940 cagcaacgtt ggatgtgtat aacactgttt tgtattgtag atgtactctt tttatataag 33000 gagaagttta aaatagaaat tacgataacg taaagtgggt ttttaccggc taaatttgaa 33060 cgttttccct ttccttccct ggtcctgtac ttgcagtatt gcactgtcgc ctgtcggttc 33120 atcgatcgga acgacgacgt cgtcgcgcgc ggcactatgg gcactagtac tttggtggca 33180 gtggcacacg ttcgtagtgt gtgtcacttt ggtgtctgga gcctgctgga gcaagacagc 33240 aagtcaggct gaggccggct actcagctgg gcgagtgggt gttgctgcga gctttggcgg 33300 atatcagggt atgcatatat ggcaacggct ccaaaaacgc gctgctgcga ctgcgatgcc 33360 ctgcagcatt gcaatggctg gccggccggt gcgcgttgct tggaagaaag atgctgtccg 33420 gtcggcgaac cagcctcgat cagccatgcg gttgtgatgc atgcgcatgc ttaacagtct 33480 ggaccctaac acgggctggg tgaagctgaa gaggacctaa tttttggtag ttttgtttga 33540 ctgaattatt ggtagttaag ctagattagt cctacttttg ggccggggct ggctttctgg 33600 cctagctcaa cacataattg tttttttttc tccatacgga cggcgtacat gcgttcaaat 33660 tttaaacctt gaccttttac ttttattcac gtacggtgta ctgaacccgc cgtagtccgg 33720 tagaaaaaag tttaaaaatg agagacacga cctcatgtta accgtcaccc gtgcatcatc 33780 gatggacgta cgttgacagc atcacaacga ctacaaaaat atcaaataat gcatgttaat 33840 ctgattctta gtatagcctt tcggtgtcgg caaattgcaa tgccgtgtgc cactatgttc 33900 attctcaaaa aaactgacca attgacgccg gactaatgct ggatcaacaa cgaccgcaca 33960 agtcgtgata gtaacggtct agctagtttt cacttttcag agattaattt agagacagct 34020 agctagctca ttagttagtt agcaaattag ctagttattt gttagttagc taatttcact 34080 aatatttttt agccaactaa ctatatatat cttcagtgca ttcaaacagt ccctatataa 34140 tctagattaa tttagttgta gctatttggc atcctagatt aatggaactc agattttcat 34200 cattagacaa cactgtccac ggtcgtattt cttcattatt tatgatactg tagcagtttg 34260 taggtacata aaaacgttga acatgttcaa acacattaaa aataaatatt tcatacacat 34320 tttgtgccca tatcatacta catttgaaat taaattacat tcttctcgca aataatacat 34380 tgacaatttt atctagaaaa gtattcgcgg ttccctcttc atctcccaat ccggtatcgt 34440 gatgacttga tggtatttaa tcgttgtcgt cttcatcaca tcaatcaaat agttcttccc 34500 gtatatgtta ctacctcaaa tgaaaaaatt attccttcaa cattggataa cttggtaagt 34560 ccaataaaat tatgaatttc atcttcgaga cagcgaaaga ttgctcaata tgataagtgc 34620 aattggttga atacatctca tcatcacatg tcagtcaatt aattagtcga tcaacgatga 34680 aaattaagcc ctgaggttca ccgtgctaca aaacgacatg attgtgtgta cagttttcgt 34740 actatttaag ttttacctcc ttcctatatg aaacataatt cctcaacgat tgtttgcttc 34800 ggattaaagc ggactattta gattggtgta ggtgtaatct ataaaaacaa aagtactata 34860 51tt gaaccagtag aaaccggtat agattaaagt caagggaacg actgaaagtc cacaaagcta 34920 ctggattcca tgagcttctg tagataacaa gcccgacatg gcgacgtcca catgggccgg 34980 gtctgaaaat tcccttccca gcaaataaag agaaactagc aaattgttca tatatgctac 35040 ggttctattt atacttatgt atagaatata aaatataaag atatgagtgt attgttagtt 35100 atgtggatat gaatgtgagt ttagagctaa taatcatagt tcgaatctct atttgtatat 35160 aattttagca tttttataat ttaaatgtga ggtatacgat gaaaatcata aaactaggaa 35220 aattagtgtt ttaatatagt acagatatta ttctctgaac caaagacaca caccagaata 35280 caactcacat atgcattgca cgtgtgaagc tccgacacac agggcgcaac aactcagctg 35340 ctttctttac gtgatcagat tgctagagta cttcacaaag ggacgccgag agagtatcgt 35400 tcaccaacct ggcagtggca gttgccgcat tgtactagtg tacaaaagcc cagctgaacg 35460 gtgatcagtg gcctcccaat gccattctgt gtaggcgatc acagatctat caacaccacc 35520 aaacccaagc cagcacactg caaaacacca aaaggattga agcaagctgc caaatggcac 35580 gcgttgcagg aacaccgacc ctgcctgatt cagagtcaga aatgcaatta tagacgagtc 35640 tatgggcatt gtcactgaca gaatgacagg gcaaagatac cacagtttca cgcgaaaaca 35700 tggccgtggc acgttggccg ctcgaggcaa caacttctat attggcatag ccagaatgat 35760 aatattgttg tacaactcag ttagagatcg agtcgcacga tcatgctcgc actctctgat 35820 tagggacgta acaatctgtt ggttaataag ttctcctttc ataccacata ataaatagtc 35880 aaagaaccga tacgcgaacc aacattcata ccgagattcg atctaatatc taccggccat 35940 cttttcgact ccccgaacca tagccggagt ggccatgggc atttctccgc ccttgatcag 36000 catggcatct ccttccaatt ctggaggtac aaatctatgt tctatgattt ctaatggcta 36060 ggattacgag tcgaggtctt aaaatctaat agtggtatca aagccatggt tttaggctcg 36120 ggctctaact ataaatgaca gttttcctta agttttatga caaaatccga gaaaaacacc 36180 tcttaactct ttgtattcgt gttcctactc acgtagaaca agcaaaaggg gagaaagaaa 36240 ctctaaccca acatttccac ctttcccaaa ctctaatcct aaggcagtcc ataatctgag 36300 cacccaagca cacaaagaaa gggaaagcag acgttaggaa agcagccatg tgctatatat 36360 tcgaagacct agcctacaca cacatctgtg aaactgtatt aaattgttaa atatttttag 36420 tttgttaaat atttttaaat tacgttaact cgtcttttct agttattact tatagcttca 36480 aatctagttt catttacgat aaagttcaat aatcattatc cttctcttga ttactatttt 36540 catttggtat gaatcatgat cggttgtttt tctctaaatg tttgggtgag taatctttag 36600 gcttgacaaa cgaggatggt gaattcattt tttacgcatc tcatattatt taaagtgcat 36660 cacttgtaca cgagcacaaa ctcgatggca tgtaagtgat tttagggtgt gattgacaca 36720 caaaataggc tattgggagt gacaaacaac tcctcaagtc taaggactaa ataagaatat 36780 ttagaaaatt agtatatttg gatatctcat ttaaaaaccc attgaagctt gatttttggt 36840 taataacatc ctacattatt tttagggcct gttttgggaa ctagagatgt tctaagggct 36900 accactcaat tggataccta attagttgtt gactaatctt gattagttgg atgacagaaa 36960 atagaatgga actaggtaac taagggggtg ttggattaca ctagagataa tagttagctg 37020 ctaaaattag ctgaagacat ccaaacactt tagctaatag ttaaactatt agctattttt 37080 agcaaattag ctaatactcc ctccgatttt tttatttgac gtttgttagt gaaaatctga 37140 actattgagt gtcaaataaa taaaaatgga ggtagtagtt aggtagacat ttgttaggta 37200 gctaattaca ctaacaatgt ttagccaact aactattagt tctagtgcat taaaacatcc 37260 tctgaggtga tgacctgcat gtggtatgac ttaagtcact tggcctcatt agcctaattt 37320 gcaactctac atgcagccat cgtcctagag ttaattggtc actagttagt tgctaactgg 37380 tcatagttag ttgtccgtgt gtgctagggt ttctttatac catatagata ggctttatcg 37440 aaaaaagata tttatttaaa cacgatctaa actcatagat tctcatatcg cacaattata 37500 tttttttatt tttaaattat ttgtatatat atttacaata tcaattctta ggcacaaatg 37560 aacatgtggt atactggtta gagactcttt gttttgtctc tcactgagtg agcaggattt 37620 gaaccctcat aggctgcgtt ggacgcacgg ccgcgagagt tagacgatcc acgataaaac 37680 tcatgtttta agggtgtgtt tggttgtaat gatgggaatg agacagaatg aaatgtgatg 37740 atcctcaaat aaggttgttt agtttagggt caagggttgg aacaagactg tcccagtatt 37800 gtcccagtta tccctcgaaa ttagagagac gagaggggac gcgaggaaac gtctctgacc 37860 tggctatccc tatgtgtacc gcaaccaaac gcaccataaa ttaatgatga tgatgcttat 37920 tgttgttgaa agggttgcaa ccttaactac agcactaacc gccttatcca aggatcaaac 37980 acaaaaggcc aaacctgtga agatttactg ttgtattaca taattacaag attaccctgg 38040 aatgtttcgc atgtctgtaa accgaagaaa aatagttggt tccaaacatc ccaggtacta 38100 gcctgtgtgc accttggcgc gcagaggctg gagttgattt ttcctttatc gaaaaggaaa 38160 tatttctaag cctctaacca tttttgcatt ctatgcagtg tcacctcatc agagagttaa 38220 tagttaatca agcctcccaa aaccttcatt tattaatgag ccagtgaaac tacatgatac 38280 catcatagta agaagttcca tgagtaacag gtcagcagat tcatttttca tttgtgaaac 38340 51uu tagattaaac aaaatattcc tttgggttga agtttttttt gtctatttga cttttgggtt 38400 gtaacatgaa caaaaatata ccacctgcaa ctgaaacatt caaagattca aaggtacctt 38460 cctttggaat gtttacaagc tcaaatgcct ggagggtctc tgcagtgagg ccattgccct 38520 cacttcctaa cactaagcac agagattcat tcaacatcga atcagctagt tctttggaca 38580 gcgagtagat ttttttggat gcagcactac tactttctgg atggcctgcc atcatcttca 38640 taccatactt tgtcatcaat gcatgcaggt catgccaggt accagagaca ataggaagct 38700 gcaaaggggc tccacgggct gcacgaacag cctttccatt gaaaggacca caacaagccg 38760 gaagaaggaa tactccatcc tgatggcatt tgggttcaga ggttgttaag aggtgtagag 38820 ttggaacagc aaaaactaga gtgtttggtt ttctctattg gagattgtta gtttctatgg 38880 tgcctaagat cgattcctaa gtccctaata ctaatccgta acagaaagta aaacctcgat 38940 gacgagtaga tctgatctac tgagatcaat agggaaacac acttttgttg ttgttgttgg 39000 agcgttgtcg cagttgccgt cttcataccg ttgccctgca gagaagcagc aggcatcgga 39060 ttcgagccgc tgtgggttgt agcgtttcca ggcactccga cgcttaggtg atggggcctc 39120 tggggactag ggttttgggg cgaggtacta gtgttagtct ttcctgcgac ctttagtcct 39180 atatttatag cgttgtgtgc ccggaggctc caaccgaggt taacgtggcc cctctcaatc 39240 aagggtccgt ttaaggagat agatagttgg gattagccca atctgatcac tgatgaccag 39300 ctatagagga cacacccaac agagataact aatacatata aatcatgtat tgtgtatcct 39360 ataaaaaata ttgtgtaccg gaatcagaga taaggaggta aggacatcca tcctatctta 39420 cttaagagca gaatcaaatt ctttttagca aaggagaact ggagcacaat gaaatttagt 39480 ggcattcctc aaattctatg gtagagtagc aagattattt aacactatgc aatgcagcaa 39540 taatacagtc tgacatactt caataagcgg ccttcagaaa agaatatggc atacccattt 39600 gaaagcacaa gctgatctta tcagtgttcc gaggttacca gggtcctgca aggtaggggc 39660 accagtgcac catgcttacc atgagaccca ctgaacggat atgcataact acatttgctt 39720 caaacaaaaa cagcatgcat agattgaatt atgccttgca ttctactccg tccgttcttt 39780 tttatttgtc acggtttatt agtttagttc aaaaatgaac tagcgggcga caaatattcg 39840 agaatggata tagtattatt taaggatgac agcaatcatc tctctgcaca atggtggcct 39900 tgatgggtac tgcatatcct cacctgaatc ccatcaagga ctagaatcct ctttggacaa 39960 ctgaacaacc catcaagagc atcccatcct catggctcct gaggtcgcgg aaatggttag 40020 gcatgtgcat gacagcaatc gcctcggtgg aatcagccga ttgcatcccg gacaccttct 40080 tcatcaccgc gtcgctgcaa tacacgacat taaaggactc gcgcagcacc tccgggacct 40140 aggataagca agtaatcgat gacaaggggt agaagcacag gtttaggaag agaagaaacc 40200 tcgcaggaca tgtaaatatg acaagggcct ggagttgcag aggagtacag gatgggcacg 40260 aggccgacaa aatttgacca tcacatttgg tatgtttggt taactttcgc atcatatagg 40320 ataggagtat aggactagga tccatttcaa attatcgtgc atttcataat ggtttctgaa 40380 gctttggtca ccaagtattc ctatttctcg gctggaattt cacgtttgcg ctgcgaggga 40440 gtgtttcgtc ttcgactctt cgttcagaca gaccccgcgc ggactggagc caagccgctg 40500 ctacctgccg cacgctgcca ctgtcaggtc cgctcggcac atagcgacac agcgagcgca 40560 aacatttcgt tcgcactggt ttagcaaccg aaacgctcct ctatctaatc tcttatatca 40620 tcccctattt catacttatc tctacaaaca gtgttatata tagttccacg tcatatattt 40680 tccactctac tagaaacatg tttgattcgc ccctgactgt gccacaattt acaatttatc 40740 taaggttagt aaaaaaaagt tagataaagt atgataagat ggcgaactaa atatacccta 40800 gcgacacaac agcttggaac aacttcaacc agccctagtt agccactcgt agcttcatgg 40860 taaagatttt ggataatttt ttacaagttt acacacttca tcacgacaaa gattgtgaat 40920 gctggatgtt tttggatttt gacaccttta cacacttcat aacgtgtgcc atctattaga 40980 ggagaaacga gaactgcacg cagcaacgca ccaggctaat aagtgacaac cgaacaagga 41040 aaggcgggaa gggggcaacc tggaaaatag agagcagagc agaactttat tcctcctctc 41100 aggaagtgtc cttgtcactc taaacgctaa cagccgaagg aaagaaggct gagagtgaga 41160 tttggagcca caaaaagata accggcatac tcagaactac atgggtccaa atttgagatg 41220 gtattcaaat tttagatggc aaggtctctt aggcacagct cattcgtctc ccattgcaag 41280 ttctagcgcg taatcatctc tttcctgaaa aacaaggagt ccattttatc tcagtgaagt 41340 gtctaataac ctataatcac actttgcaga tcagaaagta ttgttactca ccactggccc 41400 tcgtgcaaag tatctcccga attggagcca atatacctgc aaattgacaa acaacatgct 41460 ttgaagagcg aatttcctac tacatataga gctagatatt aattaatttc aaaaggtgtt 41520 ttagactacg tgcttgcctc atcttcatct tgagtctcga cttcaacatc gccagatggg 41580 aaaccaacac acaacaaagc atagccctga aaaggaaacc atacaataga actctaaaat 41640 ccttaagtac catacttgat aggcaacatc ggacaatgct cctttccact ttagctatga 41700 atttggattt atccgaattt atagctaagt gtctactatt ttaggacgaa gatagtactt 41760 tataattggt agctaatttg tattgatgaa catttctatg ttgcttggcc aagaataact 41820 51vv #
gatcaataaa aaaatctatt gtcatatctg taacacaaaa aatacaaaat aaattgtggg 41880 tcacattgca atcataatcc ttccttaaaa gtcgacataa tagttatgca tcaaactcta 41940 gcaattgccc aggaacattc aggaaattgt acaaactagg taactgacta aactggagga 42000 caggttaaga gtgaaataca acctctcatt tccagaccat gtacacaggg aacaataata 42060 tgtcatgggc acaatactcc aatcgtgctt caactagtgg aagaaaaaat ggatgtacct 42120 tatcttttag ttctgcagat atcccaagtg cttcaggctg ccttatctgt cctgatttta 42180 ttcggactgc acagcttgtg cagcaacctg attaccaaaa tgagttttca attgttatat 42240 agaccacacg gaacaatcaa ataagagatg ctatccaatc aaaaggtatt catagttaaa 42300 taatatatca aaagccaatg gcatgggtag tttttttttt aatttatagt tgctataagt 42360 atttgacaaa tagaaagagt cccaaatgag tccaacatgg ttctgactac atattccaaa 42420 atcaaaacca acatcacact cccacacgat actatctata tcaacataaa ggaggatgag 42480 aaatagccaa cattcagttt gcacatattg gagcagctag tggtggagct tggccataaa 42540 aagaggacgg accattatga tatgaagtgt aaagagaagg gagacagatt agtatttcat 42600 cagttccgcc actcaagaca gggcttcatc aggctctatt tgtaacaaat gaagacggtt 42660 caagctgaag ggtgacattc atgcttagca cttagtgtag ctgctactaa ctaggcatgc 42720 ataatgagtc gcccaatagc ttactgtcat gtacggccgc acttacaggc gccgtcgtga 42780 cgccaggagg gctgcagatc gcgcagccaa ggcaggcgcc acaatcagtg gctaagttta 42840 gaaagataag gattagtttc cctcttgcat gccttaatca taggagggat tggttacgat 42900 tagtttcctt ttcatatgcc ttaataatag gagagattgg ttaagattag tttccttttc 42960 atatgcctta atcataggaa aggttggttg aaccagaggc tatatatatg ccgtgtaata 43020 ggctgggaaa taatcaagca agatcaatta tccaaaactg ctcttctcct ttgcctctct 43080 caagccaccg gtgaggaatc cctcacggtg aaggtctaga gcgcgactac gaactgcgta 43140 gccgcggtcc agcgacgttg ggcgtcgagg cgcttgacaa cctggtatca gcgtcacgtc 43200 gatcctcacc cacccgctct tgccctccac catccccttc cacgcccacc acctccaccg 43260 accactccat caccatggct gagcccacca tcaaggacct catggagctg atgcagtctt 43320 tccaaaccga gttggctgcc gtgaaagcca ccatgaagga caagtcgtcc tcgtcgtcca 43380 ccgacggcgg cggcgagcgc gaggatcccc ctcgtgtgga tcggcccccc aggttccaga 43440 agctcgactt tccccggttc gatggcacca ccgacccgat gctcttcatc aacaaatgtg 43500 aatcctattt tcgccaacag cgcaccatgg cggaggaacg cgtgtggatg gcctcttata 43560 atctggagga cgtcgcgcag ctgtggttca tccagctgca ggacgacgag ggcacaccat 43620 cctgggggcg gttcaaggac ctcctcaatc ttcgcttcgg gccaccactc tgctcagcgc 43680 ccctgttcga gttggcggag tgtcgccgca ccgggaccgt ggaggaatac tccaaccggt 43740 tccaggccct gctgccgcgc gcgggtcgcc tcacggaggg ccagcgtgtc caactctaca 43800 caggcggtct ccttccccca ttgagccacg ttgttcgcat ccacaacccg gagacacttg 43860 atggggccat gagtctcgcc cgtcaagccg agcagctgga gttggcacgg ggaccgccac 43920 cggccgccag gggagcccct cgctctcttc ccccagcgcc ggcgcccaag cagccgctgc 43980 tcgccctccc tgcaccaccc gcgggcgcgc cccagccgcg accagagggt ccgcccttga 44040 agcggctatc accggaggaa caggcggaac ggcgccgcct cggcctctgc ttcaactgca 44100 atgagccgta caaccgcggc cacaaccgtg tctgccgccg catcttctac ctccatggcg 44160 tcgagctcac ggccgctgcc gaggaactcg taggggacga cccgcaggac ggcgcccctg 44220 tcttctccct cagggcagtc actggcatgc ccatctgcaa ttccatgcag gtgcgggcca 44280 ccctaggcgc caccaccctc ctcgcgctcc tggacaccgg ctccacgcat aacttcattg 44340 gggaggatgc cgcccgccgc accggcctgc cgatccagcc gcacctcacg gccactgtgg 44400 ccaacggtga acgtgtcacc tgcccaggag tcattcgccg cgcccaggtc atcatcgagg 44460 gcgagccatt cttcatcgac ctcttcgtca tgccgctcgc agggtacgac ctcgtcctag 44520 ggactcaatg gatggtcacc cttggtcgca tggtgtggga cttcgtcgac cgctccgtct 44580 ccttcctgca ccaaggtcgc caggtctcct ggtcggacgt cgccgaccgt cagcatcccc 44640 tgctcgccgc taccacgaca ccgagcgccc tccttgagga gctcttgcac tccttcgacg 44700 gggtgtttgc tgagccggcg ggtctgcccc cacagcgagc tcgggaccac gccatcgtcc 44760 tcaagccagg ctctacacct gtggcggtcc ggccgtaccg ctacccggtg gcgcacaagg 44820 atgagttgga gcggcaatgt gccgccatgt tgacccaagg catcgtacac cgcagtgact 44880 cggcgttctc ctcaccggtt ctgctggtca agaagcacga cggggcttgg cggttttgcg 44940 tggactaccg cgcccttaat gccctcaccg tcaaagacgc cttccccatc cccatcgtcg 45000 acgagctcct tgacgagctg catggtgcgc agttcttcac gaagctggac cttcgctccg 45060 gtgatagtcg cctagagggg gggtgaatag ggcgaaactg aaatttacaa atataaacac 45120 aactacaaga cggggttagc gttaggaata agaaacgagt ccgcaagaga gggcgcaaaa 45180 caaatcccaa gcgaataagc aagtgagaca cggagatttg ttttaccgag gttcggttct 45240 ctcaaaccta ctccccgttg aggaggccac aaaggcctgg tctctttcaa cccttccctc 45300 lww tctcaaacga tccacggatc gagtgagctt tctcttctca atcacttgga acacaaagtt 45360 cccacaagga ccaccacaag attggtgttt cttgccttaa ttacaagtga gtttgattgc 45420 aaagaaggat caagaaagaa gaaagcaatc caagcgcaag agctcgaaag aacacgagta 45480 aatcactctc tctagtcact atggcgttgt gtggaatttg gagaggattt gatctctttg 45540 gcgtgtctag aattgaatgc tagagctctt gtagtagttg ggaagtggaa aacttggatg 45600 caatgaatgg tggggtggtt ggggtattta tagcctcaac caccaaaagt ggccgttgga 45660 aggctgtctg ttcgatggcg caccggacag tccggtgcac accggacagt ccggtgcccc 45720 ctgccacgtc atcactgtcg ttggattctg accgttggag cttctgactt gtgggcccgc 45780 ctgggtgtcc ggtgcacacc ggacaggtac tgtttgctgt ccggtgtgcc agcatgggcg 45840 tttctgactc ctgcgcgcgc agagcgcgca ttaaatgcgc ggcagagagc cgttggcgcg 45900 gagaagaccg ttgctccgga gacgcaccgg acagtccggt gaattatagt ggactagccg 45960 ttggggtttc ccgaagctgg cgagttcctg aggccgacct cctttggcgc accggacact 46020 gtccggtgta caccggacag tccggtgaat tatagcgcga gtgcctctgg aaattcccga 46080 aggtggcgag tttgagtctg agtccccctg gtgcaccgga cagtccggtg cgccagacca 46140 ggggtgcctt cggttgcccc tttgctcttt tgttgaatcc aaaactgggt ctttttattg 46200 gctgagtgtg aaccttttac acctgtgtaa tctatacact tgggcaaact agttagtcca 46260 attatttgtg ttgggcaatt caaccaccaa aattaattag ggactaggtg taagcctaat 46320 tccctttcaa tctccccctt tttggtgatt gatgccaaca caaaccaaag caaatataga 46380 agtgcataat tgaactagtt tgcataatgt aagagtaaag gttgcttgga attgagccaa 46440 tgtaaatact tacaagatat gcagggattg tttctttctt atatattatt ttggaccacg 46500 cttgcaccac atgttttgtt tttgcaaatt ctttttgtaa atccatttca aagatctttt 46560 gcaaatggtc aaaggtaaat gaataagagt ttgcaaagca ttttcaagat ttgaaatttt 46620 ctccccctgt ttcaaatgct tttcctttga ctaaacaaaa ctccccctaa aagagatcct 46680 cctcttagtg ttcaagaggg ttttgatata tcatttttga aatactactt tctccccctt 46740 ttgaacacaa taggatacca attgataaat actcttggaa aacactaagt ttttgaaatt 46800 ggttgtggtg cggtcctttt tgctttgggc tcatactttc tccccctttg gcatgaatcg 46860 ccaaaaacgg aatcattaga gcctatcgaa gtactatcgt cccctttggt cataagtaaa 46920 tgagttaaga ttataccaaa gacgaaatcc ggtcttttag ctttgggttc ttactctctc 46980 cccaaagaca aggtccttta ttggagcgat ggcgaaggat gagttaccga gtggaagcct 47040 ttgtctttca ccgaagactc caattccctt tcaatatacc tatgacttgg tttgaaatag 47100 acttgaaaac acattagtca tagcatatat gattaaaagt ataaatgagc tatgtgtgca 47160 atctagcaaa agaagttgcg tgaatcaaga atattgagct catgcctaag tttggtaaaa 47220 gtttgttcat caagaggctt ggtaaagata tcggctaatt gatctttagt gttaatgtaa 47280 gaaatctcga tatccccctt ttgttggtga tccctaagaa aatgataccg aatggctatg 47340 tgcttagtgc ggctatgctc gacgggattg tcggtcattt tgattgcact ctcattatca 47400 catagcaaag ggactttggt taatttgtaa ccgtagtccc gcagggtttg cctcatccaa 47460 agcaattgcg cgcaacaatg acctgcggca atgtactcag cttcggcggt ggaaagagcg 47520 accgaatttt gcttctttga agcccaagac accaaagatc ttcccaagaa ctggcaagtc 47580 cctgatgtgc tcttcctatt aattttgcac cccgcccaat cggcatccga ataaccaatc 47640 aaatcaaatg tggatccccg agggtaccaa agtccaaact taggagtata agccaaatat 47700 ctcaagattc gttttacggc cgtaaggtgg gattccttag ggtcggattg gaatcttgca 47760 cacatgcata cggaaagcat aatgtccggt cgagaagcac ataaatagag taaagaacct 47820 atcatcgacc ggtatacctt ttgatccacg gacttacctc ccgtgtcgag gtcgagatgc 47880 ccattggttc ccatgggtgt cttgatgggc ttggcatcct tcattccaaa cttgcttaga 47940 atatcttgag tatactttgt ttggctaagg aaggtgccct cttggagttg tttgacttgg 48000 aatcctagaa aatacttcaa ctcccccatc atagacatct cgaatttctg tgtcatgatc 48060 ctactaaact cttcacatgt agactcgtta gtagacccaa atataatatc atcaacataa 48120 atttggcata caaacaagtc attttcaaga gttttagtaa agagtgtagg atcggccttg 48180 ccgactttga agtcattagc aataaggaaa tctctaaggc attcatacca tgctcttggg 48240 ggcttgcttg agcccataaa gcgcctttga gagcctatag acatggttag ggtactcact 48300 atcttcaaag ccgggaggtt gctcaacata gacctcttcc ttgattggtc cgttgaggaa 48360 ggcacttttc acgtccattt gataaagctt aaagccatgg taagtagcat aggctaataa 48420 tattcgtatt gactcaagcc tagctacggg tgcataggtt tcaccgaaat ccaaaccttc 48480 ' gacttgggag tatcccttgg ccacaagtcg tgctttgttc cttgtcacca caccatgctc 48540 atcttgcttg ttgcggaaga cccatttggt ccctacaaca ttttgggtta ggacgtggaa 48600 ccaaatgcca tacctcattc ctcgtgaaat tgttgagctc ctcttgcatc gcccaccacc 48660 caatccgaat ctgaagtgct tcctctatcc tatgtggctc aatagaggaa acaaaagagt 48720 aatgctcaca aaaatgggca acacgagatc gagtagttac ccccttatga atgtcgccga 48780 51xx ggatggtgtc gacggggtga tctcgttgta ttgcttggtg gactcttggg tgtggcggtc 48840 ttggttcttc ctcatgctcc ttctcttgat catttgcatc tcccccttga tcattgtcat 48900 tatcttgagg tggctcatct tcttgatttt gcccttcatc aacttgagcc tcatcctcat 48960 tttgagttgg tggagatgct tgcgtggtgg aggatgattg atcttgtgca cttggaggct 49020 cttcggattc cttaggacac acatccccaa tggacatgtt ccttagcgct atgcatggag 49080 cctcttcatt acctatctca tcaagatcaa cttgctgtac ttgagagccg ttagtctcat 49140 caaacacaac gtcacatgag acttcaacta gtccagtgga cttgttaaag accctatatg 49200 cccttgtgtt tgagtcataa ccaagtaaaa agccttctac agttttagga gcaaatttag 49260 attttctacc tcttttaaca agaataaagc atttgctacc aaaaactcta aagtatgaaa 49320 tgttgggctt tttaccggtt aggagttcat atgatgtctt cttgaggatt cggtgaagat 49380 ataaccggtt gatggcgtag caggcggtgt tgaccgctgc ggcccaaaac cggtccggtg 49440 tcttgtactc atcaagcatg gttcttgcca tgtccaatag agttcgattc ttcctctcca 49500 ctacaccatt ttgttgaggg gtgtagggag aagagaactc atgcttgatg ccctcctcct 49560 caagaaagcc ttcaatttgt gagttcttga actccgtccc gttgtcgctt cttattttct 49620 tgatccttaa gccgaactca ttttgagccc gtctcaagaa tcccttcaag gtctcttggg 49680 tttgagattt ttcctgtaaa aagaataccc aagtgaagcg agaataatca tccacaataa 49740 ctagacagta cttactcccg ccgatgctta tgtaagcaat cgggccgaat agatccatgt 49800 gtaggagctc cagtggcctg tcggtcgtca tgatgttctt gtgtggatga tgagctccaa 49860 cttgcttccc cgcctgacat gcgctacaaa tcctgtcttt ctcaaaatga acatttgtta 49920 atcctaaaat gtgttctccc tttagaagct tatgaagatt cttcatccca acatgggcta 49980 gtcggcggtg ccagagccaa cccatgttag tcttagcaat taagcatgtg tcgagttcag 50040 ctctatcaaa atctactaag tatagctgac cctctaacac acccttaaat gctattgaat 50100 cattacttct tctaaagaca gtgacaccta catcagtaaa aagacagttg tagcctattt 50160 gacataattg agaaacagaa agcaagttgt aatctaaaga atcaacaaga aaacattgga 50220 aatagaatgg tcaggagata tagcaatttt accaagtcct ttgaccaaac cttgatttcc 50280 atccccgaat gtgatagctc gttggggatc ttggtttttc tcatatgagg agaacattct 50340 tttctcccca gtcatatggt ttgtgcaccc gctgtcgagt atccaacttg agcccccgga 50400 tgcataaacc tacaaaacaa atttagttct tgactttagg tacccaaact gttttgggtc 50460 ctttggcatt agaaacaaga actttgggta cccaaacaca agtcttggaa cccttgtgtt 50520 tgcccccaac aaacttggca actactttgc cggatttgtt agtcaaaaca taggatgcat 50580 caaaagtttt aaatgaaata gcatgatcat ttgaagcatt aggagttttc tttctaggca 50640 acttagcacg ggttggttgc ctagaactag atgtctcacc cttatacata aaagcatggt 50700 tagggccaga gtgagacttc ctagaatgag ttctcctaat tttgctctca ggataaccgg 50760 cagggtacaa aatgtaaccc tcgttatcct gaggcatggg agccttgccc ttaacaaagt 50820 tggacaagtt cttaggaggg gcattaagtt tgacattgtc tcccctttgg aagccaatgt 50880 catccttaat gccggggcgt ctcccattat aaagcatgct acgagcaaat ttaaatttct 50940 cattctctaa gttgtgctcg gcaattttag catctagttt tgttatatga tcattttgtt 51000 gtttaattaa agccatatga tcatgaatag catcaatatc aacattttta catctagtgc 51060 aaatagtgac atgctcaatg gtggatgtag atggtttgca agaattaagt tcaacaatct 51120 tagcacgaag tatatcattc ttatctctaa gatcagaaat tgtaattttg caaacatcaa 51180 aatctttagc cttagcaatt aaattttcat tttctaatct aaggctagca agagatacat 51240 tcaattcatc aatcttaaca agcaaatcaa cattatcatc tctaagattg ggaattgaaa 51300 catcaccaaa tatgtgaatc aaccttaa 51328 <210> 107 <211> 58 <212> DNA
<213> Zea mays <400> 107 cactgtgcgt cctctgcagg cagttgttga catgagcgca tcgtcactgc tgaatcgc 58 51yy

Claims (2)

CLAIMS:

1. A maize chromosomal target site located within a maize plant cell, on chromosome 5, wherein said target site is flanked 5' by nucleotides 5,454 to 25,454 of SEQ
ID NO: 106 and flanked 3' by nucleotides 25,513 to 45,513 of SEQ ID NO: 106, wherein the target site comprises a heterologous nucleic acid comprising a gene of interest.

2. A method of making a transgenic maize plant comprising inserting a heterologous nucleic acid on chromosome 5, wherein said inserted heterologous nucleic acid is flanked 5' by nucleotides 5,454 to 25,454 of SEQ ID NO: 106 and flanked 3' by nucleotides 25,513 to 45,513 of SEQ ID NO: 106.